High Fidelity Restriction Endonucleases

ABSTRACT

Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer.

CROSS REFERENCE

This is a divisional of U.S. patent application Ser. No. 13/022,561,filed Feb. 7, 2011, which is a continuation-in-part of U.S. patentapplication Ser. No. 12/172,963 filed Jul. 14, 2008, herein incorporatedby reference. This application also claims priority from U.S.provisional application Ser. No. 60/959,203 filed Jul. 12, 2007, Ser.No. 61/301,666 filed Feb. 5, 2010, and Ser. No. 61/387,800 filed Sep.29, 2010, herein incorporated by reference.

BACKGROUND

Restriction endonucleases are enzymes that cleave double-stranded DNAsin a sequence-specific manner (Roberts, R. J. Proc Natl Acad Sci USA102: 5905-5908 (2005); Roberts, et al. Nucleic Acids Res 31:1805-1812(2003); Roberts, et al. Nucleic Acids Res 33:D230-232 (2005); Alves, etal. Restriction Endonucleases, “Protein Engineering of RestrictionEnzymes,” ed. Pingoud, Springer-Verlag Berlin Heidelberg, New York,393-407 (2004)). They are ubiquitously present among prokaryoticorganisms (Raleigh, et al., Bacterial Genomes Physical Structure andAnalysis, Ch. 8, eds. De Bruijin, et al., Chapman & Hall, New York,78-92 (1998)) in which they form part of restriction-modificationsystems, which mainly consist of an endonuclease and amethyltransferase. The cognate methyltransferase methylates the samespecific sequence that its paired endonuclease recognizes and rendersthe modified DNA resistant to cleavage by the endonuclease so that thehost DNA can be properly protected. However, when there is an invasionof foreign DNA, in particular bacteriophage DNA, the foreign DNA will bedegraded before it can be completely methylated. The major biologicalfunction of the restriction modification system is to protect the hostfrom bacteriophage infection (Arber Science 205:361-365 (1979)). Otherfunctions have also been suggested, such as involvement in recombinationand transposition (Carlson, et al. Mol Microbiol, 27:671-676 (1998);Heitman, Genet Eng (NY) 15:57-108 (1993); McKane, et al. Genetics139:35-43 (1995)).

The specificity of the approximately 3,000 known restrictionendonucleases for their greater than 250 different target sequencescould be considered their most interesting characteristic. After thediscovery of the sequence-specific nature of the first restrictionendonuclease (Danna, et al., Proc Natl Acad Sci USA 68:2913-2917 (1971);Kelly, et al., J Mol Bio/51:393-409 (1970)), it did not take long forscientists to find that certain restriction endonucleases cleavesequences which are similar but not identical to their definedrecognition is sequences under non-optimal conditions (Polisky, et al.,Proc Natl Acad Sci USA, 72:3310-3314 (1975); Nasri, et al., NucleicAcids Res 14:811-821 (1986)). This relaxed specificity is referred to asstar activity of the restriction endonuclease.

Star activity is a problem in molecular biology reactions. Star activityintroduces undesirable cuts in a cloning vector or other DNA. In casessuch as forensic applications, where a certain DNA substrate needs to becleaved by a restriction endonuclease to generate a unique fingerprint,star activity will alter a cleavage pattern profile, therebycomplicating analysis. Avoiding star activity is also critical inapplications such as strand-displacement amplification (Walker, et al.,Proc Natl Acad Sci USA, 89:392-396 (1992)) and serial analysis of geneexpression (Velculescu, et al., Science 270:484-487 (1995)).

SUMMARY

In an embodiment of the invention, a method is provided of identifying afidelity index (FI) of a restriction endonuclease and variants thereofthat includes selecting a reaction buffer and a DNA substrate containingthe binding and cleavage site of the restriction endonuclease;permitting the serially diluted restriction endonuclease or variantsthereof to cleave the DNA substrate; and determining an FI for each ofthe restriction endonucleases and the one or more variants thereof.

In an embodiment, the method further comprises comparing the FI for therestriction endonuclease and the variants thereof to obtain animprovement factor of, for example, greater than 2 for the variant.

In an embodiment of the invention, a buffer is selected that includespotassium acetate, Tris acetate and magnesium acetate; or magnesiumchloride.

Additional embodiments include:

-   -   (a) A composition, comprising: an enzyme comprising SEQ ID No. 1        in which one or more amino acids have been mutated, wherein the        position of one or more mutations is selected from the group        consisting of S36, K77, P154, E163, Y165 and K185.    -   (b) A composition, comprising: an enzyme comprising SEQ ID No. 2        in which one or more amino acids have been mutated, wherein the        position of one or more mutations is selected from the group        consisting of K198 and Q148.    -   (c) A composition, comprising: an enzyme comprising SEQ ID No. 3        in which one or more amino acids have been mutated, wherein the        position of one or more mutations is selected from the group        consisting of S15, H20, E34, M58, Q95, R106, K108, T181, R187        and R199.    -   (d) A composition, comprising: an enzyme comprising SEQ ID No. 4        in which one or more amino acids have been mutated, wherein the        position of one or more mutations is selected from D16, D148 and        E132.    -   (e) A composition, comprising: an enzyme comprising SEQ ID No. 5        in which one or more amino acids have been mutated, wherein the        position of one or more mutations is selected from the group        consisting of K75, N146 and D256.    -   (f) A composition, comprising: an enzyme comprising SEQ ID No. 6        in which one or more amino acids have been mutated, wherein the        position of one or more mutations is selected from the group        consisting of E198 and D200.    -   (g) A composition, comprising: an enzyme comprising SEQ ID No. 7        in which one or more amino acids have been mutated, wherein the        position of one or more mutations is selected from the group        consisting of K229, E025, R034 and Q261.    -   (h) A composition, comprising: an enzyme comprising SEQ ID No. 8        in which the position of the mutation is K225.    -   (i) A composition, comprising: an enzyme comprising SEQ ID No. 9        in which one or more amino acids have been mutated, wherein the        position of one or more mutations is selected from the group        consisting of H137, D177, K363, K408, R411, Q215, Q226 and Q230.    -   (j) A composition, comprising: an enzyme comprising SEQ ID No.        10 wherein the position of the mutation is F376.    -   (k) A composition, comprising: an enzyme comprising SEQ ID No.        11 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of R78, T140, E152, R199 and F217.    -   (l) A composition, comprising: an enzyme comprising SEQ. ID No.        12 in which one or more amino acid have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of G26, P105, T195, Q210, Y147, Y193, K114, T197,        S245, D252 and Y027.    -   (m) A composition, comprising: an enzyme comprising SEQ ID No.        13 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of H10, N208, K48, K74, R75, Y56, K58 and M117.    -   (n) A composition, comprising: an enzyme comprising SEQ ID No.        14 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of K014, Q069, E099, R105, R117, G135 and Y035.    -   (o) A composition, comprising: an enzyme comprising SEQ ID No.        15 in which one or more amino acids have been mutated, wherein        the position of one or more mutations are selected from the        group consisting of N106, Q169, E314 and R126.    -   (p) A composition, comprising: an enzyme comprising SEQ ID No.        16 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of T20, P52, Y67, K68, R75, E86, Q90, S91, Q93, H121        and G172.    -   (q) A composition, comprising: an enzyme comprising SEQ ID No.        17 in which one or more amino acids have been mutated, wherein        the position of one or more mutations selected from the group        consisting of E059, P065, S108, N172, K174, Q179, G182 and Y055.    -   (r) A composition, comprising: an enzyme comprising SEQ ID No.        18 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of N212 and L213.    -   (s) A composition, comprising: an enzyme comprising SEQ ID No.        19 having a mutation at position N65.    -   (t) A composition, comprising: an enzyme comprising SEQ ID No.        20 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of E007, D011, E049, R073, R114, G137, S210 and R213.    -   (u) A composition, comprising: an enzyme comprising SEQ ID No.        21 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of P079, E086, H096 and E218.    -   (v) A composition, comprising: an enzyme comprising SEQ ID No.        22 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of E32, S081, G132, F60 and S61.    -   (w) A composition, comprising: an enzyme comprising SEQ ID No.        23 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of G013, G016, K018, P052, R053, K070, E071, D072,        G073, S84, E086, R090, K094, R095, P099, P103, K113, N135, S151,        P157, G173, T204, S206, K207, E233, N235, E237, S238, D241,        K295, S301 and S302.    -   (x) A composition, comprising: an enzyme comprising SEQ ID No.        24 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of S64, S80, S162, T77/T96 and N178.    -   (y) A composition, comprising: an enzyme comprising SEQ ID No.        25 in which the position R232 is mutated.    -   (z) A composition, comprising: an enzyme comprising SEQ ID No.        26 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of S50, Y81, N93 and W207.    -   (aa) A composition, comprising: an enzyme comprising SEQ ID No.        27 having a mutation at G26.    -   (bb) A composition, comprising: an enzyme comprising SEQ ID No.        28 having a mutation at E112/R132.    -   (cc) A composition, comprising: an enzyme comprising SEQ ID No.        29 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of N016, S33, P36, H76, P87, N89, R90, T138, K141,        K143, Q221, Q224, N253, Q292, R296, T152, G326 and T324.    -   (dd) A composition, comprising: an enzyme comprising SEQ ID No.        30 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of K024, P214, E146, N251 and Y095.    -   (ee) A composition, comprising: an enzyme comprising SEQ ID No.        31 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of G075, Q099, G155, P022 and R90.    -   (ff) A composition, comprising: an enzyme comprising SEQ ID No.        32 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of 5097 and E125.    -   (gg) A composition, comprising: an enzyme comprising SEQ ID No.        33 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of K021, I031 and T120.    -   (hh) A composition, comprising: an enzyme comprising SEQ ID No.        34 in which one or more amino acids have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of K7, T10, N11, N14, Q232 and T199.    -   (ii) A composition, comprising: an enzyme comprising SEQ ID No.        35 in which one or more amino acid have been mutated, wherein        the position of one or more mutations is selected from the group        consisting of P92, P144, G197 and M198.

Any of the above compositions may be further characterized in that themutated enzyme has an FI in a predetermined buffer that is greater thanthe enzyme without the mutations in the predetermined buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the comparison of PvuI-HF and PvuI-WT activity.

In FIG. 1A, the asterisk (*) sign denotes the lane on the left (lane 2)in which star activity is no longer detected. The number (#) signdenotes the lane on the right (lane 8) in which partial digestionoccurs. The starting concentration of the PvuI-WT was calculated to be77 units.

In FIG. 1B, complete digestion was observed until lane 15, after whichstar activity was observed. The window of dilution allowing for completedigestion expanded from 6 dilutions to 15 dilutions in the series. Thestarting concentration of the PvuI-HF was calculated to be at least 9600units.

FIGS. 2A and 2B show the comparison of HindIII-HF and HindIII-WTactivity.

In FIG. 2A, the asterisk (*) sign denotes the lane on the left (lane 9)in which star activity is no longer detected. The number (#) signdenotes the lane on the right (lane 15) in which partial digestionoccurs. The starting concentration of the HindIII-WT was calculated tobe 9,600 units.

In FIG. 2B, complete digestion was observed until lane 13, after whichstar activity is observed. The window of dilution allowing for completedigestion expanded from 6 dilutions to 13 dilutions in the series. Thestarting concentration of the HindIII-HF was calculated to be at least2,400 units.

FIGS. 3A and 3B show the comparison of DraIII-HF and DraIII-WT activity.

In FIG. 3A, the asterisk (*) sign denotes the lane on the left (lane 12)in which star activity is no longer detected. The number (#) signdenotes the lane on the right (lane 12) in which partial digestionoccurs. Neither star activity nor partially digested DNA was observed.The starting concentration of the DraIII-WT was calculated to be 1,200units.

In FIG. 3B, complete digestion was observed until lane 12, after whichstar activity is observed. The starting concentration of the DraIII-HFwas calculated to be at least 1,200 units.

FIGS. 4A and 4B show the comparison of KpnI-HF and KpnI-WT activity.

In FIG. 4A, the * sign denotes the lane on the left (lane 9) in whichstar activity is no longer detected. The # sign denotes the lane on theright (lane 13) in which partial digestion occurs. In FIG. 1A, thestarting concentration of the KpnI-WT was calculated to be 2,000 units.

In FIG. 4B, complete digestion was observed throughout with no staractivity or partial digestion. The starting concentration of the KpnI-HFwas calculated to be greater than 12,000 units.

FIGS. 5A-5B shows the comparison of StyI-HF and StyI-WT.

In FIG. 5A, the * sign shows the beginning of the star activity on itsleft (lane 6), the # sign shows the beginning of partial activity on itsright (lane 12). The starting amount of Sty-WT was calculated to be1,000 units.

In FIG. 5B, star activity was observed in the first 2 lanes and partialdigestion from lane 14 or 15. The starting amount of StyI-HF wascalculated to be 4,000 units.

FIG. 6 shows a comparison of BglI-HF and BglI-WT on pXba. The BglI-HFhas an FI of at least 8,000 while the BglI-WT has an FI of 32, providingan improvement factor of at least 250. The right panel is thetheoretical digestion pattern.

FIG. 7 shows a comparison of BsrDI-HF and BsrDI-WT on pBR322. TheBsrDI-HF has an FI of at least 1,000 in NEB4, while the BsrDI-WT has anFI of ½, providing an improvement factor of at least 2,000. The rightpanel is the theoretical digestion pattern.

FIG. 8 shows a comparison of BclI-HF and BclI-WT in NEB4 on lambda(dam⁻). The BclI-HF has an FI of at least 2,000, while the BclI-WT hasan FI of 32, providing an improvement factor of at least 64. The rightpanel is the theoretical digestion pattern.

FIG. 9 shows a comparison of BglII-HF and BglII-WT on pXba. The BglII-HFhas an FI of at least 32,000, while the BglII-WT has an FI of 16,providing an improvement factor of at least 2,000. The right panel isthe theoretical digestion pattern.

FIG. 10 shows a comparison of BstEII-HF and BstEII-WT on lambda DNA. TheBstEII-HF has an FI of at least 2,000, while the BstEII-WT has an FI of4, providing an improvement factor of at least 500. The right panel isthe theoretical digestion pattern.

FIG. 11 shows a comparison of SfiI-HF and SfiI-WT on pBC4. The SfiI-HFhas an FI of at least 8,000 in NEB4, while the SfiI-WT has an FI of 64,providing an improvement factor of at least 120. The right panel is thetheoretical digestion pattern.

FIG. 12 shows a comparison of SmaI-HF and SmaI-WT on pXba. The SmaI-HFhas an FI of at least 256,000, while the SmaI-WT has an FI of 64,providing an improvement factor of at least 4,000. The right panel isthe theoretical digestion pattern.

FIG. 13 shows a comparison of BsmBI-HF and BsmBI-WT on lambda DNA. TheBsmBI-HF has an FI of 250 in NEB4, while the BsmBI-WT has an FI of 4,providing an improvement factor of at least 64. The right panel is thetheoretical digestion pattern.

FIG. 14 shows a comparison of BstNI-HF and BstNI-WT on pBR322. TheBstNI-HF has an FI of 500 in NEB4, while the BstNI-WT has an FI of 4,providing an improvement factor of at least 120. The right panel is thetheoretical digestion pattern.

FIG. 15 shows a comparison of MluI-HF and MluI-WT on lambda DNA. TheMluI-HF has an FI of at least 32,000 in NEB4, while the MluI-WT has anFI of 32, providing an improvement factor of at least 1,000. The rightpanel is the theoretical digestion pattern.

FIG. 16 shows a comparison of NspI-HF and NspI-WT on pUC19. The NspI-HFhas an FI of 500 in NEB4, while the NspI-WT has an FI of 32, providingan improvement factor of at least 16. The right panel is the theoreticaldigestion pattern.

FIG. 17 shows a comparison of BsrFI-HF and BsrFI-WT on pBR322. TheBsrFI-HF has an FI of at least 500 in NEB4, while the BsrFI-WT has an FIof 16, providing an improvement factor of at least 32. The right panelis the theoretical digestion pattern.

DETAILED DESCRIPTION OF EMBODIMENTS

The generation of mutants of restriction endonucleases with improvedspecificity for a single sequence is not straightforward. Numerousproblems were encountered. These include the following: a mutated enzymehad reduced or no activity, did not have reduced star activity oractually had increased star activity. Alternatively, a mutated enzymecould not be cloned and therefore could not be analyzed.

Failure to produce a mutant resulted from any of a variety of possiblecauses including any of the following. It could be due to failedinverted PCR. It is also possible that the mutation which generated newspecific activity was toxic to a host cell even if it expressed thecognate methylase under conditions that were normally protective forexpression of the non-mutated restriction endonuclease. In thesecircumstances, no viable mutant clone would be obtained. Alternatively,the mutant might have a preference for a particular buffer such thatwhen tested in another buffer, no activity would be detected. Anotherdifficulty encountered, was that although generally a crude lyzate ofeach mutation was tested, in some case, the enzyme had to be purified todetect activity where activity was not detected in the lysate scoringthe assay negative.

It was surprising to note that in several examples, a change of aproline to an alanine resulted in variants with a desired FI of at leastgreater than 250 and yielding an improvement factor of at least twofold. This was exemplified in variants of PvuI, BamHI, NruI and SpeI.

Other challenges in producing high fidelity mutants include the size ofthe DNA encoding some restriction endonucleases. This DNA may bedifficult to amplify by PCR given the large size of the template.Moreover, the PCR products in some circumstances did not readilytransform into a new host. Even if a host cell transformation wassuccessful, transformed is cells did not always produce colonies andhence could not be readily detected. In some cases, even if the colonieswere obtained from transformation, they could be not cultured in anycondition.

Reasons for reduction in the specific activity of mutants may resultfrom any of the following: the mutation interferes with the folding ofthe protein which significantly lowered the expression level or themutation affects the specific enzyme activity.

For example, this was observed for StyI mutants: N34A, F35A, D58A, F65A,K66A, K67A, F100A, N148A, E213A, F250A, T251A, D258A, D262A, N283A,R293A, F294A, R295A, R296A, D298A, D299A, M304A, M310A, D318A, S337A,S346A and F371A.

Loss of enzyme activity may result from causes that include any of thefollowing: the mutation deleted the residues which are important incatalysis; or the mutations changed residues that are important infolding, thus, the misfolded mutant protein is inactive.

For example, this was observed for StyI mutants M33A, D37A, F41A, D55A,D71A, N77A, R79A, E80A, F81A, T82A, E83A, F97A, F101A, E136A, W137A,M138A, M140A, K144A, Q145A, R151A, R255A, R259A, S261A, T264A, F278A,R281A, T284A, M297A, H305A, N306A, D314A, D338A and E382A.

Generating high fidelity mutants requires painstaking work. Multiplemutants are selected and tested and only a relatively small number showhigh fidelity. It was not possible to predict by extrapolation whichmutants are likely to show improved properties.

Examples of assays performed to identify high fidelity variants ofrestriction endonucleases are shown in FIGS. 1-17. The figures show theresults in a single buffer for both wild type and high fidelityvariants. All the figures show amounts and types of cleavage of DNAafter a series of two fold dilutions from left to right on the gel withthe concentration of enzyme decreasing in the direction of the triangle.Table 1 details the results for the 33 exemplified enzymes. Therestriction endonuclease reaction buffers (buffers 1-4) used in theexamples are defined for example in the NEB catalog (2009/10). Otherbuffers may be selected according to the preference of the user.

The assays yield an FI that is the ratio of the highest restrictionenzyme concentration not to show apparent star activity as determined bythe presence of bands associated with star activity to the restrictionenzyme concentration that completely digests 1 μg of standard DNAsubstrate in 50 μl reaction for 1 hour at a defined temperature in astandard NEB buffers. In FIGS. 6-17, a box is placed in the figures toshow star activity bands. In embodiments of the invention, the FI is forexample preferably at least 250 for example greater than 500 for examplegreater than 1000, for example, greater than 5000.

A fidelity improvement value is calculated as a ratio of the FI of thevariant divided by the FI of the non-mutant enzyme. In an embodiment ofthe invention, the improvement value is for example preferably at least2, for example, at least 4, for example, at least 8, for example, atleast 16.

In one embodiment, the FI refers to the ratio of the highest restrictionenzyme amount not to show apparent star activity to the amount thatcompletely digests 1 μg of standard DNA substrate in 50 μl reaction for1 hour at specific temperature in standard NEB buffers.

TABLE 1 Summary of properties of HF enzymes SEQ Enzyme Sub FI1′ FI2′FI3′ FI4′ Example ID No. PvuI-HF pXba ≧2000 (⅛) ≧16000 (1) ≧4000 (¼)≧16000 (1) 1 1 HindIII-HF λ ≧260000 (½) ≧260000 (½) ≧250 ( 1/2000)≧520000 (1) 2 2 DraIII-HF λ ≧120 ( 1/16) ≧1000 (½) ≧32 ( 1/64) ≧2000 (1)3 3 KpnI-HF pXba ≧1000000 (1) ≧1000000 (1) ≧30000 ( 1/500) ≧1000000 (1)4 4 StyI-HF λ ≧4000 (½) 2000 (1) ≧16 ( 1/250) 4000 (½) 5 5 BsaJI-HFpBR322 ≧1000 (¼) ≧4000 (1) ≧4000 (1) ≧4000 (1) 6 6 BsaWI-HF pXba 8 (1/64) 120 (1) ≧120 (1)) ≧4000 (1) 7 7 BglI-HF λ ≧4000 (½) ≧8000 (1) ≧500( 1/16) ≧8000 (1) 8 8 BsrDI-HF pBR322 ≧120 (⅛) ≧500 (1) ≧64 ( 1/16)≧1000 (1) 9 9 NsiI-HF pXba ≧250 ( 1/32) ≧1000 (⅛) ≧500 ( 1/16) ≧8000 (1)10 10 DpnII-HF λ(—) 4000 (¼) 2000 (⅛) 64 ( 1/128) 8000 (1) 11 11 BclI-HFλ(—) ≧250 ( 1/32) ≧500 (¼) ≧32 ( 1/64) ≧2000 (1) 12 12 BglII-HF pXba≧8000 (⅛) ≧128000 (1) 2000 (½) ≧32000 (¼) 13 13 BstEII-HF λ ≧64 ( 1/32)≧1000 (½) ≧32 ( 1/64) ≧2000 (1) 14 14 BanII-HF λ(—) ≧4000 (1) ≧2000 (½)≧500 (⅛) ≧2000 (½) 15 15 PspGI-HF pBC4 ≧1000 (¼) ≧4000 (1) ≧4000 (1)≧4000 (1) 16 16 SpeI-HF T7 ≧4000 (½) ≧250 (⅛) ≧120 (½) ≧1000 (1) 17 17BsmAI-HF FX174 ≧4000 (1) ≧2000 (½) ≧500 (⅛) ≧4000 (1) 18 18 BstXI-HF λ≧500 (½) ≧1000 (1) ≧500 (½) ≧1000 (1) 19 19 SfiI-HF pBC4 250 (½) ≧1000(⅛) ≧32 ( 1/250) ≧8000 (1) 20 20 PmeI-HF pXba ≧2000 (⅛) ≧500 ( 1/16) ≧32( 1/250) ≧8000 (1) 21 21 SmaI-HF pXba ≧2000 ( 1/500) ≧32000 ( 1/32) ≧32( 1/32000) ≧256000 (1) 22 22 AatII-HF pXba NC NC NC ≧1000 (1) 23 23ApoI-HF pXba ≧2000 (½) ≧4000 (1) ≧1000 (¼) ≧2000 (½) 24 24 BsmBI-HF λ 32(½) 120 (½) ≧120 (½) 250 (1) 25 25 BmtI-HF pXba 25600 (¼) 25600 (¼) 2000( 1/500) 1000000 (1) 26 26 BstNI-HF pBR322 ≧120 (½) ≧500 (1) ≧120 (¼)500 (1) 27 27 MluI-HF λ ≧16000 (½) ≧32000 (1) ≧2000 ( 1/16) ≧32000 (1)28 28 BanI-HF λ ≧1000 (½) ≧250 (⅛) ≧250 (⅛) ≧2000 (1) 29 29 KasI-HFpBR322 ≧8000 (½) ≧16000 (1) ≧2000 (⅛) ≧16000 (1) 30 30 NruI-HF λ ≧64 (1/250) ≧1000 ( 1/16) ≧100 ( 1/16) ≧16000 (1) 31 31 NspI-HF pUC19 ≧4000(1) 500 (1) ≧250 (⅛) 500 (1) 32 32 BsrFI-HF pBR322 ≧500 (1) ≧64 (⅛) >100≧500 (1) 33 33

Diluent (Dil) A, B and C and Buffers 1-4 are defined in the NEB catalog2009/10 page 87.

EXAMPLES Example 1 Engineering of High Fidelity (HF) PvuI 1. Expressionof PvuI

PvuI was expressed in E. coli transformed with pUC19-PvuIR andpACYC184-PvuIM, each containing PvuI endonuclease and methylase genes.The cells were grown at 30° C. overnight in LB with Amp and Cam.

2. Mutagenesis of PvuI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 8, 11, 12, 16, 17, 20, 21, 22, 23,26, 28, 29, 30, 31, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48, 49, 53, 55,57, 59, 61, 63, 65, 66, 67, 69, 70, 71, 72, 73, 77, 78, 80, 81, 82, 87,88, 90, 92, 93, 96, 97, 101, 102, 104, 106, 107, 108, 109, 110, 111,115, 116, 119, 120, 121, 122, 126, 127, 129, 131, 132, 135, 138, 139,144, 146, 147, 148, 150, 151, 152, 154, 155, 157, 158, 160, 161, 162,163, 167, 169, 170, 172, 173, 174, 178, 180, 182, 183, 184, 185, 186,187, 189, 192, 194, 195, 196, 201, 202, 203, 205, 206, 210, 211, 214,215, 218, 219, 220, 221, 226, 230, 231, 232, 233, 235, 236, 238, 239,240, 241, 246, 247, 248, 249, 251, 253, 254; while Tyr was changed toPhe at positions 18, 52, 56, 84, 91, 130, 143, 165, 204, 242.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI-digestion. The treated product was then transformed into E. colistrain ER2683.

3. Selection of PvuI-HF

Selection of PvuI-HF was achieved using comparison of activity in NEB3and NEB4 (New England Biolabs, Inc., Ipswich, Mass. (NEB) using pXba DNAas substrate. PvuI-WT has more activity in NEB3. The one with moreactivity in NEB4 was selected. 6 mutants were found to have moreactivity in NEB4: S36A, K77A, P154A, E163A, Y165F and K185A. P154A hadmuch higher activity than WT in NEB4. Normally, the one with highestactivity in NEB4 was the one with improved star activity. PvuI(P154A)was designated as PvuI-HF. This is the first time that an effectivemutation was a Proline to Alanine mutation.

4. Purification of PvuI-HF

Two liters of cell ER2683(pUC19-PvuI(P154A), pACYC184-PvuIM)) were grownin LB with 100 μg/ml Amp and 33 μg/ml at 30° C. for overnight. The cellswere harvested and sonicated in 20 ml 10 mM Tris-HCl, pH7.5, 50 mM NaCl.After a centrifugation at 15,000 rpm for 30 minutes, the supernatant wasloaded on the 5 ml HiTrap™ Heparin HP column (GE Healthcare, now Pfizer,Inc., Piscataway, N.J.) pre-balanced by the same buffer by syringeinjection. The column was then loaded on the system by the followingprocedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100 ml 10 mMTris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed by a 10 ml10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions were thentested for activity. The fractions with highest activity were furtherconcentrated by Vivaspin® 15R (Vivascience, now Sartorius VivascienceGmbH, Goettingen, Germany) and stored in glycerol at −20° C.

5. Comparison of PvuI-HF and PvuI-WT

The FIs of PvuI-HF and PvuI-WT have been determined separately on pXbaDNA in four NEB buffers with diluent B. The comparison is shown in FIG.1, and the result is listed in Table 2 (below).

TABLE 2 Comparison of PvuI-HF and PvuI-WT PvuI-HF PvuI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1 12.5% ≧2000  6.3% 32 ≧64 NEB2 100% ≧16000   25% 32 ≧500 NEB3   25% ≧4000  100% 32 ≧125 NEB4  100%≧16000 12.5% 32 ≧500 PvuI-HF performed best in NEB2 and NEB4, in whichthe FI was ≧16,000; WT PvuIperformed best in NEB3, in which the FI was32. So the overall improvement factor was ≧16,000/32 = ≧500.

Example 2 Engineering of HF HindIII

HindIII recognizes and digests at A/AGCTT as described in Example 21 ofInternational Publication No. WO 2009/009797. A mutant HindIII(K198A)was selected as the HF version of the HindIII. Further characterizationof this mutant revealed that though the performance of HindIII(K198A) onone hour scale was excellent, it did not perform well in the overnightdigestion. While searching for more mutants, HindIII(Q148A) was alsofound to be partially good. A further step toward greater improvementwas to change the Alanine to all other amino acid residues. Among those,HindIII(Q148I) was found to be excellent in both one hour and overnightreaction, and designated to be HindIII-HF (FIG. 2).

The HindIII-HF was expressed in ER3081 (pUC19-HindIIIR(Q148I)M). Thegrowth and purification methods were performed according toWO/2009/009797.

The following table (Table 3) compares the FIs of HindIII-HF andHindIII-WT.

TABLE 3 Comparison of HindIII-HF and HindIII-WT HindIII-HF HindIII-WTImprovement Buffer Activity FI Activity FI Factor NEB1   50% ≧260000 25% 32 ≧8000 NEB2   50% ≧260000 100% 250 ≧1000 NEB3 0.05% ≧250  25%4000 ≧ 1/32 NEB4  100% ≧520000  50% 32 ≧16000 The HindIII-HF had thebest activity in NEB4; the FI of HindIII-HF in NEB4 was ≧520000; the WTHindIII had the best activity in NEB2. The FI of HindIII-WT in NEB2 was250. So the overall improvement factor was ≧2,000.

Example 3 Engineering of HF DraIII 1. Expression of DraIII

DraIII recognizes and digests at CACNNN/GTG. DraIII was expressed in E.coli ER3081 with pAGR3-DraIIIR( ) and pACYC-DraIIIM( ). The cells weregrown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of DraIII

The length of DraIII protein is 227 amino acids. Total 132 amino acidsites of DraIII protein were initially designed to be mutated into Alais (or Phe). Cys, Asp, Glu, Phe, His, Lys, Met, Asn, Gln, Arg, Ser, Thr,Gly and Trp were mutated to Ala. Try was mutated to Phe. These were: 7,9, 10, 11, 12, 14, 15, 16, 17, 18, 20, 21, 22, 23, 28, 29, 31, 32, 34,35, 37, 40, 42, 43, 44, 45, 47, 51, 54, 55, 57, 58, 59, 60, 64, 65, 66,67, 68, 72, 73, 74, 76, 77, 82, 83, 84, 88, 89, 90, 91, 93, 94, 95, 96,99, 101, 102, 104, 106, 107, 108, 111, 112, 113, 114, 115, 117, 120,121, 123, 124, 127, 128, 130, 136, 137, 138, 139, 140, 141, 142, 144,145, 146, 147, 150, 154, 155, 156, 157, 158, 160, 161, 165, 167, 169,170, 171, 172, 173, 175, 176, 180, 181, 183, 184, 185, 187, 189, 190,192, 193, 196, 198, 199, 200, 201, 202, 205, 207, 208, 209, 211, 212,213, 214, 216, 217, 218, 219, 22, and 223.

The point mutagenesis of the selected mutations was done by inverse PCR.The PCR reaction in a reaction volume of 100 μl, contained 2 μl of eachPCR primer, 1 μl pAGR3-DraIIIR, 400 μM dNTP, 4 units of Deep Vent™ DNApolymerase (NEB), and 10 ul 10× Thermopol buffer with additional water.

The PCR reaction conditions were 94° C. for 5 min, followed by 25 cyclesof 94° C. 30 sec, 55° C. 60 sec, 72° C. 4 min and a final extension timeis at 72° C. for 7 mins. The PCR product was digested by 20 units ofDpnI for 1 hour. The digested product was transformed into E. coliER3081(pACYC-DraIIIM).

3. Selection of DraIII-HF

Four colonies of each mutation were grown up in LB with Amp and Cam at37° C. overnight. The standard cognate and star activity assays ofDraIII were performed using pXba as substrate in NEB4 buffer and 10%glycerol.

The mutants S15A, H20A, E34A, M58A, Q95A, R106A, K108A, T181A, R187A,R199A, N202D, T181G, T181N, T181Q, T181C, T181V, T181L, T181I, T181M,D55A, D55S, D55C, D55G, D55N, T12A, H20A, E34A, H45A, T57A, M58A, T60A,S66A, R76A, F90A, M94A, T101A, C115A, F169A, N172A, R173A, H189A, N193Aand Q95A/K104A were picked out in screening assays. After several roundsof comparison in different conditions and substrates, DraIII(T181A) wasfound to be a preferred mutant, retaining high cleavage high activity,but displaying substantially reduced star activity. DraIII (T181A) waslabeled DraIII-HF.

4. Comparison of DraIII-HF and DraIII-WT

The DraIII-WT and DraIII-HF (T181A) proteins were purified using Heparinand Source 15S Column. The assay condition for detailed comparison wasas follows: NEB4 (or NEB1, 2, 3), 37° C., 1 h; 2 μl purified protein in20 μl reaction system; lambda DNA as substrate. The comparison is shownin FIGS. 3A and 3B, and the result is listed in Table 4.

TABLE 4 Comparison of DraIII-HF and DraIII-WT DraIII-HF (T181A)DraIII-WT Improvement Substrate Activity FI Activity FI factor Buffer16.25% ≧120  16% 16 ≧8 Buffer2   50% ≧1000 100% 2 ≧500 Buffer3 1.56% ≧32 50% 2 ≧16 Buffer4  100% ≧64000  50% 0.5 ≧128000 DraIII-HF has mostactivity in NEB4, in which the FI was at least 64,000; the DraIII-WT hasmost activity in NEB2, in which the FI is 2. The overall FI improvementfactor was at least 32,000 fold.

Example 4 Engineering of HF KpnI

KpnI recognizes and digests at GGTAC/C as described in Example 26 ofInternational Publication No. WO 2009/009797. A triple mutantKpnI(D16N/E132A/D148E) was selected as the high fidelity version of theKpnI. While D148E and E132A were introduced by site-directedmutagenesis, the D16N was introduced by PCR. Further characterization ofthe mutations in this triple mutant revealed that the removal of theE132A will further improve the restriction enzyme, especially in theaspect of the enzyme specific activity. The triple mutantKpnI(D16N/E132A/D148E) has a specific activity of 200,000 units/mgprotein, while KpnI(D16N/D148E) has a specific activity of 1,800,000units/mg protein. The double mutant is 9 times more active than theprevious triple mutant, so the double mutant KpnI(D16N/D148E) wasdesignated as the KpnI-HF.

The KpnI-HF was expressed in ER2523(pAGR3-KpnI(D16N/D148E),pSYX20-KpnIM). The growth and purification methods were performedaccording to WO/2009/009797.

The following table (Table 5) compares the FIs of KpnI-HF and KpnI WT.

TABLE 5 Comparison of KpnI-HF and KnpI-WT KpnI-HF KpnI-WT RelativeRelative Improvement Buffer Activity FI Activity FI factor NEB1  100%≧1,000,000 100% 16  62,500 NEB2  100% ≧1,000,000  25% 16  62,500 NEB3 0.2%   ≧30,000  6%  8  3,750 NEB4  100% ≧1,000,000  50%  4 250,000 TheKpnI WT had the best activity in NEB1, the FI of KpnI-WT in NEB1 was 16;the KpnI-HF had the best activity in NEB1, NEB2 and NEB4. The FI ofKpnI-HF in these three buffers were all highest at ≧1,000,000. Theoverall improvement factor was ≧62,500.

Example 5 Engineering of HF StyI 1. Expression of StyI

StyI recognizes and digests at C/CWWGG. StyI was expressed in E. coli(ER2833) with pACYC-StyIM and placzz1-StyIR. The cells were grown at 37°C. overnight in LB with Amp and Cam.

2. Mutagenesis of StyI

The point mutagenesis of the selected mutations was done by inverse PCR.237 amino acid mutations were made in StyI as follows: Cys, Asp, Glu,Phe, His, Lys, Met, Asn, Gln, Arg, Ser, Thr, Trp were mutated to Ala.Tyr was mutated to Phe. These were at the positions: 7, 9, 10, 11, 12,14, 16, 22, 23, 24, 25, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 42, 43, 49, 51, 52, 53, 54, 57, 58, 59, 61, 62, 64, 65, 66, 69, 70,73, 75, 76, 78, 79, 80, 81, 82, 85, 91, 92, 93, 95, 96, 97, 98, 99, 100,102, 103, 104, 105, 106, 109, 111, 112, 114, 116, 118, 119, 122, 123,124, 125, 126, 128, 129, 130, 131, 135, 136, 137, 139, 140, 141, 142,143, 144, 145, 146, 147, 150, 151, 152, 153, 155, 157, 158, 159, 163,164, 165, 166, 167, 170, 172, 173, 175, 176, 177, 178, 181, 183, 187,188, 192, 193, 194, 195, 196, 200, 203, 204, 205, 207, 209, 211, 212,213, 214, 216, 218, 219, 220, 221, 222, 227, 229, 230, 232, 234, 235,236, 237, 238, 239, 241, 242, 245, 247, 248, 249, 250, 252, 253, 254,256, 257, 258, 259, 260, 261, 263, 266, 267, 269, 272, 274, 277, 280,282, 283, 284, 286, 288, 289, 291, 292, 293, 294, 295, 296, 297, 298,303, 304, 305, 307, 308, 309, 313, 317, 318, 319, 320, 323, 324, 326,327, 329, 331, 335, 336, 337, 339, 340, 343, 345, 346, 347, 349, 350,351, 353, 355, 356, 359, 360, 361, 363, 365, 366, 368, 369, 370, 372,373, 376, 377, 379, 381, and 382.

The method of primer design and PCR can be performed as described inpublished PCT application WO 2009/0029376 (Example 1). The PCR productwas digested with DpnI and transformed into competent ER2833(pACYC-StyIM).

3. Selection of StyI-HF

Four colonies of each mutation were grown up in LB with Amp and Cam at37° C. overnight. The cognate activity assay and star activity assays ofStyI were performed using lambda in NEB4 and ExoI buffer and 20%glycerol respectively.

The mutants K75A, N146A and D256A were picked out in screening assays.After several rounds of comparison in different conditions andsubstrates, K75A was found to be the preferred mutant, retaining highcleavage high activity, but displaying substantially reduced staractivity. StyI(K75A) was labeled StyI-HF.

4. Comparison of StyI-HF and StyI-WT

The comparison of StyI-HF and StyI-WT in NEB4 is shown in FIGS. 5A and5B, and the result is listed in Table 6.

TABLE 6 Comparison of StyI-HF and StyI-WT StyI-HF StyI-WT RelativeRelative Improvement Buffer Activity FI Activity FI factor NEB1   50%≧4000  25% 32  ≧125 NEB2  100%  2000 100% 16   125 NEB3  0.4%  ≧16  50%32   ≧0.5 NEB4   50%  4000  25% 16   250

StyI-WT and StyI-HF had the most activity in NEB2. The FI for StyI-WTwas 16 and for StyI-HF was 2000. The overall FI improvement factor was125.

Example 6 Engineering HF BsaJI 1. Expression of BsaJI

BsaJI was expressed in E. coli transformed with pRRS-BsaJIR+M, whichcontains BsaJI endonuclease and methylase gene in same plasmid. Thecells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of BsaJI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thr,Phe, Trp, were changed to Ala at positions 9, 10, 14, 17, 18, 19, 20,22, 23, 24, 27, 30, 32, 35, 39, 42, 43, 48, 50, 51, 52, 53, 55, 56, 57,60, 61, 65, 66, 67, 68, 70, 71, 72, 73, 78, 79, 81, 83, 84, 86, 87, 88,90, 91, 92, 94, 95, 99, 101, 103, 104, 106, 110, 111, 113, 114, 117,119, 120, 121, 123, 127, 129, 131, 132, 134, 136, 138, 140, 141, 142,147, 152, 153, 157, 158, 159, 162, 163, 165, 166, 167, 169, 170, 175,178, 181, 183, 184, 185, 186, 187, 188, 189, 194, 196, 197, 198, 199,200, 202, 203, 204, 206, 211, 212, 213, 214, 215, 216, 218, 220, 222,225, 226, 227, 228, 229, 230, 231, 233, 238, 239, 240, 241, 246, 247,249, 250, 251, 252, 253, 254, 255, 257, 260, 262, 265, 267, 268, 269,270, 271, 273, 274, 276, 277, 280, 281, 282, 283, 285, 287, 288, 290,291, 293, 294, 295, 298 and 299; while Tyr is changed to Phe at thepositions of 21, 59, 62, 77, 89, 105, 130, 191, 208, 272, 286 and 296.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER3081.

3. Selection of BsaJI-HF

Selection of BsaJI-HF was achieved using comparison of activity in NEB3and NEB4 using pBR322 DNA as substrate. E198A and D200A have highestactivity. D200A has much lower star activity than WT in NEB4. BsaJI(D200A) is designated as BsaJI-HF.

4. Purification of BsaJI-HF

Two liters of cell ER3081 (pRRS-BsaJIR(D200A)+M) were grown in LB with100 μg/ml Amp, 33 μg/ml Cam and 0.5 mM IPTG at 37° C. for overnight. Thecells were harvested and sonicated in 50 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions arethen test for activity. The fractions with highest activity were furtherconcentrated by Amicon® Ultra 30 KDa (Millipore, U.S.A; now Merck,Germany). The concentrated BsaJI-HF was then added same volume ofglycerol and stored at −20° C.

5. Comparison of BsaJI-HF and BsaJI-WT

The FIs of BsaJI-HF and WT BsaJI have been determined separately onpBR322 DNA in four NEB buffers with diluent A. The result is listed inTable 7.

TABLE 7 Comparison of BsaJI-HF and BsaJI-WT BsaJI-HF BsaJI-WTImprovement Buffer Activity FI Activity FI Factor NEB1  25% ≧1000 100%64  ≧15 NEB1 100% ≧4000 100% 64  ≧60 NEB3 100% ≧4000  25% 16 ≧250 NEB4100% ≧4000 100% 64  ≧60 BsaJI-HF performed best in the NEB2, 3 and 4, inwhich the FI was ≧4000; WT BsaJI performed best in NEB1, 2, and 4, inwhich the FI was 64. So the improvement factor in NEB4 was ≧4000/64 ≧64.

Example 7 Engineering of HF BsaWI 1. Expression of BsaWI

BsaWI was expressed in E. coli transformed with pLacZZ1-BsaWIR andpACYC-MspIM, each contains BsaWI endonuclease and methylase gene. Thecells were grown at 30° C. overnight in LB with Amp and Cam and inducedat 30° C. with 0.5 mM of IPTG for 18 hours.

2. Mutagenesis of BsaWI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 9, 10, 13, 16, 17, 18, 20, 23, 24, 25,26, 28, 29, 30, 31, 34, 35, 36, 39, 42, 43, 45, 46, 48, 51, 54, 58, 60,62, 63, 64, 65, 66, 69, 70, 71, 74, 75, 78, 80, 81, 82, 84, 85, 86, 88,89, 92, 93, 96, 99, 100, 101, 102, 104, 105, 107, 109, 113, 114, 115,117, 121, 112, 123, 124, 127, 128, 129, 130, 131, 133, 136, 137, 138,140, 141, 142, 145, 149, 151, 152, 153, 154, 155, 156, 160, 163, 164,165, 166, 167, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 181,184, 189, 195, 196, 197, 200, 202, 203, 209, 210, 211, 212, 213, 214,216, 218, 219, 221, 222, 228, 229, 230, 231, 233, 234, 237, 239, 241,243, 247, 248, 250, 251, 254, 255, 258, 259, 260, 261, 264, and 266;while Tyr is changed to Phe at the positions of 11, 57, 106, 147, 157,215, 224, 236, and 265.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER3081.

3. Selection of BsaWI-HF

Selection of BsaWI-HF was achieved using comparison of activity in NEB3and NEB4 using lambda DNA as substrate. The following mutants showedchanges: K229A, E025A, R034A and Q261A. WT BsaWI can complete digestionin both buffers when grown in small culture; Q261A was noticed to onlygive a stable partial pattern. This could be due to the fact that themutant grew poorly in small culture. When grown in large culture andpurified, the partial pattern was eliminated and the substrate wasinstead digested completely, and the results also proved to be a ishigh-fidelity mutant when tested upon the substrate pXba.

4. Purification of BsaWI-HF

Two liters of cell ER3081(pLacZZ1-BwaWI(Q261A), pACYC-MspIM)) were grownin LB with 100 μg/ml Amp and 33 μg/ml at 30° C. for overnight. After 8hours, the culture was induced with 0.5 mM IPTG. The cells wereharvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl.After a centrifugation at 15,000 rpm for 30 minutes, the supernatant wasloaded on the 5 ml HiTrap™ Heparin HP column (GE Healthcare, now Pfizer,Inc., Piscataway, N.J.) pre-balanced by the same buffer by syringeinjection. The column was then loaded on the system by the followingprocedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100 ml 10 mMTris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed by a 10 ml10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions were thentested for activity. The fractions with highest activity were furtherconcentrated by Vivaspin® 15R (Vivascience, now Sartorius VivascienceGmbH, Goettingen, Germany). The concentrated BsaWI-HF was then added anequal volume of glycerol and stored at −20° C.

5. Comparison of BsaWI-HF and BsaWI-WT

The FIs of BsaWI-HF and BsaWI-WT have been determined separately on pXbaDNA in four NEB buffers with diluent A. The result is listed in Table 8(below).

TABLE 8 Comparison of BsaWI-HF and BsaWI-WT BsaWI-HF BsaWI-WTImprovement Buffer Activity FI Activity FI Factor NEB1  1.6%   8 12.5% 4   2 NEB1  100%  120   50%  8  ≧15 NEB3  3.1%  ≧250  3.1% 64  ≧4 NEB4 100% ≧4000  100% 16 ≧250 BsaWI-HF is most active in NEB2 and NEB4, inwhich the best FI is ≧4000; BsaWI-WT is most active in NEB4, in whichthe FI is 16. The overall improvement factor is ≧4000/16 = ~250.

Example 8 Engineering of High Fidelity BglI 1. Expression of BglI

BglI was expressed in E. coli transformed with pUC19-BglIR andpSYX20-BglIM, each contains BglI endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Kan.

2. Mutagenesis of BglI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 8, 12, 14, 15, 16, 17, 18, 19, 22,23, 24, 25, 27, 28, 29, 31, 34, 36, 39, 40, 43, 44, 45, 46, 47, 48, 50,52, 54, 5, 57, 60, 61, 65, 67, 68, 70, 71, 72, 73, 75, 76, 77, 78, 79,81, 84, 86, 87, 88, 91, 92, 94, 95, 96, 99, 100, 101, 102, 103, 105,107, 108, 110, 112, 113, 114, 115, 116, 117, 118, 122, 123, 124, 125,128, 130, 131, 132, 134, 135, 136, 152, 158, 159, 160, 161, 163, 164,165, 166, 167, 170, 172, 173, 174, 176, 177, 178, 179, 180, 181, 183,184, 185, 186, 187, 188, 189, 193, 194, 196, 197, 202, 203, 204, 205,208, 211, 215, 216, 221, 222, 224, 225, 226, 227, 228, 229, 230, 231,234, 236, 239, 241, 242, 243, 245, 249, 250, 251, 255, 256, 259, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 275, 276, 277, 279, 281,283, 286, 287, 289, 290, and 291; while Tyr is changed to Phe at thepositions of 19, 13, 33, 53, 66, 119, 127, 153, 199, 218, 233, 252, and258.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed is into E. colistrain ER2566.

3. Selection of BglI-HF

Selection of BglI-HF was achieved using comparison of activity in NEB4using lambda DNA as substrate. BglI-WT has low activity in NEB4, so anymutants with similar or more activity than WT in NEB4 were selected,then they were checked against glycerol for comparison of star activitylevels. Only one mutant, K225A, showed similar activity to WT in NEB4while also decreasing star activity when tested in glycerol. BglI(K225A)is designated as BglI-HF.

4. Purification of BglI-HF

Two liters of cell ER2566(pUC19-BglI(K225A), pSYX20-BglIM) were grown inLB with 100 μg/ml Amp and 33 μg/ml Kan at 37° C. for overnight. Thecells were harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated BglI-HF wasthen added to an equal volume of glycerol and stored at −20° C.

5. Comparison of BglI-HF and BglI-WT

The FIs of BglI-HF and WT BglI have been determined separately on lambdaDNA in four NEB buffers with diluent B. The comparison is shown in FIG.6, and the result is listed in Table 9 (below).

TABLE 9 Comparison of BglI-HF and BglI-WT BglI-HF BglI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1   50% ≧4000 25.0%  64  ≧62NEB1  100% ≧8000  100%  64 ≧125 NEB3  6.3%  ≧500  100% 250  ≧2 NEB4 100% ≧8000   50%  32 ≧250 BglI-HF was most active in NEB2 and NEB4, inwhich the FI was ≧8000; BglI-WT is most active in NEB3, in which the FIwas 250. The overall improvement factor was ≧8000/250 = ≧32.

Example 9 Engineering of HF BsrDI 1. Expression of BsrDI

BsrDI enzyme contains two subunits: BsrDIA and BsrDIB.

To obtain a pure BsrDIA subunit, the IMPACT (Intein-MediatedPurification with an Affinity Chitin-Binding Tag) system (NEB cat:E6901) was used for the one-step purification of BsrDIA. Briefly, theBsrDIA gene was sub-cloned into the pTXB1 vector, which was thentransformed into a competent strain containing the T7 RNA polymerase,controlled by the lac operon (NEB #ER2566). After screening andsequencing, the corrected strain was selected. Cells were grown in LBmedia with Ampicillin (100 μg/ml) at 37° C. until the OD₆₀₀ reached 0.5.Then, IPTG was added to reach a final concentration of 0.4 mM for theinduction of BsrDIA for 3 hours. Cell culture was then pelleted,resuspended in ice-cold Column Buffer (20 mM Tris-HCl, pH 8.5, 500 mMNaCl) and lysed via sonication. The resulting cell lysate was thencentrifuged to remove cellular debris. Next, the supernatant was loadedonto an equilibrated Chitin Column. After washing with the loadingbuffer, the column was incubated with cleavage buffer (20 mM Tris-HCl,pH 8.5, 500 mM NaCl and 50 mM DTT) at 4° C. overnight. Finally, theBtsI.A protein was eluted with dialysis against the storage buffer (10mM Tris-HCl pH 7.4, 0.1 mM EDTA, 1 mM DTT, 50 mM KCl and 50% glycerol).

BsrDIB subunit was expressed in E. coli transformed with pUC19-BsrDIBRand pLG-BsrDIM1M2, each contains BsrDI endonuclease and methylase gene.The cells were grown at 37° C. overnight in LB with Amp and Kam.

2. Mutagenesis of BsrDI-HF

All residues of BsrDIB including Cys, Asp, Glu, Gly, His, Lys, Asn, Pro,Gln, Arg, Ser, Thr were changed to Ala at positions 7, 11, 12, 14, 15,17, 21, 22, 25, 28, 29, 30, 33, 34, 35, 37, 40, 45, 46, 47, 51, 52, 56,58, 62, 64, 65, 67, 68, 71, 72, 74, 75, 81, 83, 90, 91, 92, 93, 99, 100,101, 106, 108, 109, 112, 113, 115, 116, 120, 122, 123, 124, 132, 133,136, 137, 138, 139, 142, 143, 144, 145, 146, 150, 155, 157, 158, 161,162, 164, 168, 170, 171, 173, 174, 176, 177, 179, 180, 182, 185, 189,190, 193, 197, 200, 202, 203, 206, 210, 213, 215, 217, 218, 221, 224,225, 226, 228, 229, 230, 232, 237, 238, 241, 242, 243, 244, 245, 246,249, 253, 258, 259, 261, 264, 265, 268, 271, 272, 273, 274, 276, 278,279, 281, 285, 287, 288, 292, 294, 295, 299, 300, 301, 306, 307, 308,312, 314, 315, 317, 318, 320, 321, 324, 325, 326, 327, 328, 331, 332,335, 337, 341, 343, 345, 347, 352, 353, 354, 355, 356, 360, 361, 362,363, 364, 370, 373, 374, 376, 380, 381, 385, 387, 389, 392, 393, 395,396, 397, 405, 406, 408, 411, 415, 418, 420, 422, 425, 426, 430, 431,432, 434, 437, 445, 446, 449, 450, 454, 455, 456, 457, 458, 459, 460,463, 465, 466, 467, 469, 470, 475, 481; while Tyr is changed to Phe atthe positions of 9, 38, 63, 87, 118, 129, 169, 178, 198, 216, 251, 286,291, 303, 357, 358, 367, 371, 402, 442, 443, 448.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2566.

3. Selection of BsrDI-HF

Selection of BsrDI-HF was achieved using comparison of star activitybetween the WT BsrDIB mixed with BsrDIA and the mutant BsrDIB mixed withBsrDIA in NEB4 on pBR322 DNA as substrate. Eight mutants are found tohave less star activity in NEB4:H137A, D177A, K363A, K408A, R411A,Q215A, Q226A, Q230A.

To further reduce the star activity, we combine the above mutations tomake double mutations: K363A/Q230A, K363A/K408A, Q230A/K408A. Then BsrDIwith mutations on BsrDIB of Q230A/K363A is designated as BsrDI-HF.

4. Purification of BsrDI-HF

Two liters of cell ER2566(pUC19-BsrDI(Q230A/K363A), pLG-BsrDIM1M2)) weregrown in LB with 100 μg/ml Amp and 33 μg/ml Kam at 37° C. for overnight.The cells were harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5,50 mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions arethen test for activity. The fractions with highest activity were furtherconcentrated by Vivaspin® 15R (Vivascience, now Sartorius VivascienceGmbH, Goettingen, Germany). The concentrated BsrDI-HF was then addedsame volume of glycerol and stored at −20° C. condition.

5. Comparison of BsrDI-HF and BsrDI-WT

The FIs of BsrDI-HF and BsrDI-WT have been determined separately onpBR322 DNA in four NEB buffers with diluent A. The result is shown inFIG. 7 and listed in Table 10 (below).

TABLE 10 Comparison of BsrDI-HF and BsrDI-WT BsrDI-HF BsrDI-WTImprovement Buffer Activity FI Activity FI Factor NEB1  12.5%  ≧120   6%1  ≧120 NEB1  100%  ≧500  100% 4  ≧120 NEB3   6%  ≧64 12.5% 4  ≧16 NEB4 100% ≧1000   25% ½ ≧2000 BsrDI-HF performed best in NEB4, in which theFI was ≧1000; BsrDI-WT performed best in NEB2 and NEB3, in which the FIwas 64. So the overall improvement factor was ≧1000/0.5 = ≧2000.

Example 10 Engineering of HF NsiI 1. Expression of NsiI

NsiI was expressed in E. coli transformed with placzz1-NsiIR andpACYC-NsiIM, each contains NsiI endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of NsiI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thr,Phe, Trp, were changed to Ala at positions 8, 9, 10, 11, 12, 13, 18, 21,22, 23, 24, 26, 27, 32, 34, 35, 42, 44, 45, 46, 47, 49, 50, 52, 53, 54,55, 57, 58, 60, 61, 69, 70, 73, 74, 79, 80, 84, 85, 87, 90, 91, 92, 93,95, 96, 97, 98, 99, 100, 102, 103, 105, 106, 108, 109, 110, 113, 114,115, 117, 118, 119, 120, 121, 122, 123, 124, 126, 134, 135, 137, 138,139, 140, 142, 144, 145, 146, 149, 151, 153, 154, 155, 156, 159, 160,161, 162, 163, 166, 167, 170, 173, 174, 175, 178, 179, 180, 181, 182,183, 184, 186, 188, 189, 190, 191, 192, 195, 197, 198, 199, 200, 201,202, 203, 206, 207, 209, 210, 211, 213, 215, 216, 217, 219, 221, 222,225, 230, 231, 232, 234, 235, 236, 237, 239, 242, 243, 244, 245, 246,249, 250, 251, 256, 257, 259, 260, 261, 263, 264, 268, 269, 271, 272,273, 276, 277, 278, 279, 281, 282, 283, 285, 287, 288, 290, 292, 294,295, 297, 298, 299, 302, 303, 306, 307, 308, 309, 310, 312, 315, 316,319, 320, 323, 325, 327, 329, 333, 334, 336, 337, 338, 340, 341, 344,347, 349, 350, 352, 353, 354, 355, 358, 359, 360, 362, 363, 365, 366,367, 371, 372, 373, 375, 376 and 377; while Tyr is changed to Phe at thepositions of 30, 40, 62, 65, 71, 76, 83, 86, 141, 226, 233, 255, 289,311, 326, 335, 351, 357, 378.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER3081.

3. Selection of NsiI-HF

Selection of NsiI-HF was achieved using comparison of activity in NEB3and NEB4 using pXba DNA as substrate. NsiI-WT has more activity in NEB3,the one with more activity in NEB4 were selected. 148 mutants are foundto have more activity in NEB4. F376A has much higher activity than WT inNEB4. Normally the one with highest activity in NEB4 is the one withimproved star activity. NsiI (F376A) is designated as NsiI-HF.

4. Purification of NsiI-HF

Two liters of cell ER3081 (placzz1-NsiI(F376A), pACYC-NsiIM)) were grownin LB with 100 μg/ml Amp, 33 μg/ml Cam and 0.5 mM IPTG at 37° C. forovernight. The cells were harvested and sonicated in 50 ml 10 mMTris-HCl, pH 7.5, 50 mM NaCl. After a centrifugation at 15,000 rpm for30 minutes, the supernatant was loaded on the 5 ml HiTrap™ Heparin HPcolumn (GE Healthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balancedby the same buffer by syringe injection. The column was then loaded onthe system by the following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl, 100 ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradientand followed by a 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The elutedfractions are then test for activity. The fractions with highestactivity were further concentrated by Amicon Ultra 30 KDa (Millipore,U.S.A; now Merck, Germany). The concentrated NsiI-HF was then added samevolume of glycerol and stored in the −20° C. condition.

5. Comparison of NsiI-HF and NsiI-WT

The FIs of NsiI-HF and WT NsiI have been determined separately on pXbaDNA in four NEB buffers with diluent A. The result is listed in Table 11(below).

TABLE 11 Comparison of NSiI-HF and NsiI-WT NsiI-HF NsiI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1   3%  ≧250  6.3% 32  ≧8 NEB112.5% ≧1000   25% 32  ≧30 NEB3   6%  ≧500  100% 32  ≧15 NEB4  100% ≧800012.5% 32 ≧250 NsiI-HF performed best in NEB4, in which the FI was ≧8000;WT NsiI performed best in NEB3, in which the FI was 32. So theimprovement factor in NEB4 was ≧8000/32 = ≧250.

Example 11 Engineering of HF DpnII 1. Expression of DpnII

DpnII was expressed in E. coli 3081 transformed with pBAD241-DpnII RM.The cells were grown at 30° C. overnight in LB with Amp.

2. Mutagenesis of DpnII

The point mutagenesis of the selected mutations was done by inverse PCR.189 amino acid mutations were made in DpnII as follows. Cys, Asp, Glu,Phe, His, Lys, Met, Asn, Gln, Arg, Ser, Thr, Trp were mutated to Ala.Try was mutated to Phe. These were: 7, 8, 9, 10, 12, 13, 14, 15, 16, 17,19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 31, 32, 33, 35, 36, 38, 40, 42,44, 45, 46, 50, 51, 52, 54, 55, 56, 57, 59, 61, 62, 63, 64, 66, 69, 76,77, 78, 80, 81, 82, 86, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 104, 105, 106, 107, 108, 109, 111, 112, 113, 116, 117, 118,120, 121, 122, 125, 126, 129, 130, 132, 135, 138, 139, 140, 141, 143,144, 145, 146, 147, 149, 150, 151, 152, 153, 156, 157, 158, 160, 161,162, 164, 168, 169, 171, 172, 173, 175, 176, 177, 178, 180, 181, 183,184, 186, 188, 189, 191, 192, 193, 195, 196, 198, 199, 200, 201, 202,205, 206, 207, 208, 211, 214, 216, 217, 218, 219, 221, 223, 224, 226,227, 228, 229, 230, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241,244, 246, 247, 248, 249, 251, 252, 254, 256, 257, 258, 259, 260, 261,262, 264, 265, 266, 267, 268, 272, 274, 275, 277, 278, 280, 281 and 282.

The method of primer design and PCR is similar to that describedpreviously. The PCR product was digested with DpnI and transformed intocompetent E. coli 3081.

3. Selection of DpnII-HF

Four colonies of each mutation were grown up in LB with Amp at 37° C.overnight. The standard screening assays of DpnII were performed usingdam⁻ lamda substrate in NEB4 buffer and 5% glycerol.

The mutants R78A, T140A, E152A, R199A, and F217A were picked out fromscreening assay. After several rounds of comparison in differentconditions and substrates, R199A was chose as candidate, retaining highcanonical enzyme activity, but displaying substantially reduced staractivity. R199A was labeled as DpnII-HF.

4. Purification of DpnII-HF

Two liters of cell E. coli 3081 (pBAD241.DpnII.RM (R199A)) were grown inLB with 100 ug/ml Amp at 30° C. for overnight. The cells were harvestedand sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl. After acentrifugation at 15,000 rpm for 30 minutes, the supernatant was loadedon the 5 ml HiTrap™ Heparin HP column (GE Healthcare, now Pfizer, Inc.,Piscataway, N.J.) pre-balanced by the same buffer by syringe injection.The column was then loaded on the system by the following procedure: 48ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100 ml 10 mM Tris-HCl, pH 7.5, 50mM-1M NaCl linear gradient and followed by a 10 ml 10 mM Tris-HCl, pH7.5, 1M NaCl step. The eluted fractions are then test for activity. Thefractions with highest activity were further concentrated. Theconcentrated Bmt-HF was then added same volume of glycerol and stored inthe −20° C. condition.

5. Comparison of DpnII-HF and DpnII-WT

DpnII-HF was 2-fold serial diluted with B and reacted in four NEBbuffers, and DpnII-WT was 2-fold serial diluted and reacted in four NEBbuffers. The result is listed in Table 12.

TABLE 12 Comparison of DpnII-HF and DpnII-WT DpnII-HF DpnII-WTImprovement Buffer Activity FI Activity FI Factor NEB1   50% 4000  25% 1 4000 NEB1   25% 2000  25%  1 2000 NEB3  0.8%  64 100% 32   2 NEB4 100% 8000  25%  1 8000 DpnII-HF performed best in NEB4, in which thepreferred FI was =8000; DpnII performed best in NEB3, where the FI was32. The overall FI improvement factor was 8000/32 = 250.

Example 12 Engineering of High Fidelity BclI 1. Expression of BclI

BclI was expressed in E. coli transformed with pRRS-BclIR andpACYC184-BclIM, each contains BclI endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp.

2. Mutagenesis of BclI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 9, 10, 11, 12, 19, 22, 23, 24, 26, 28,29, 30, 31, 35, 37, 38, 40, 42, 44, 46, 47, 49, 51, 53, 54, 55, 58, 59,62, 65, 67, 69, 72, 73, 74, 75, 76, 80, 82, 83, 85, 86, 89, 93, 94, 95,96, 97, 98, 99, 101, 103, 105, 107, 108, 109, 110, 111, 112, 113, 114,115, 120, 124, 128, 129, 130, 132, 136, 137, 138, 139, 143, 144, 145,149, 150, 151, 152, 154, 156, 160, 162, 163, 164, 166, 167, 170, 171,172, 174, 175, 178, 179, 180, 182, 183, 188, 190, 191, 195, 196, 197,199, 200, 201, 204, 205, 208, 209, 210, 212, 213, 215, 217, 218, 220,221, 222, 223, 224, 225, 226, 228, 229, 234, 235, 237, 238, 241, 243,244, 245, 249, 252, 255, 257, 260, 261, 265, 266, 267, 270, 271, 273,274, and 277; while Tyr is changed to Phe at the positions of 17, 27,36, 63, 66, 77, 87, 100, 116, 118, 133, 142, 147, 157, 192, 193, 194,207, 212, 231, 236, and 246.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2984.

3. Selection of BclI-HF

Selection of BclI-HF was achieved using comparison of activity inglycerol and NEB4 using dam− lambda DNA as the substrate. Once lowerstar activity was suspected, mutants were also compared with normalactivity in water and NEB4 on the same substrate. Mutants with similaractivity to WT in NEB4 and also with the potential to have lower staractivity were selected. 6 mutants are found to have suchcharacteristics: G26A, P105A, T195A, Q210A, Y147F, and Y193F. Severalmutants (K114A, T197A, S245A, D252A, and Y027F) showed lower activity inwater, but decreased star activity as well; they usually had higheractivity cognate activity than WT under high glycerol conditions. Onemutant showed higher activity than WT and also lower star activity:Y192F. BclI(Y192F) is designated as BclI-HF.

4. Purification of BclI-HF

Two liters of cell ER2984(pRRS-BclI(Y192F), pACYC184-BclIM)) were grownin LB with 100 μg/ml Amp at 37° C. for overnight. The cells wereharvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl.After a centrifugation at 15,000 rpm for 30 minutes, the supernatant wasloaded on the 5 ml HiTrap™ Heparin HP column (GE Healthcare, now Pfizer,Inc., Piscataway, N.J.) pre-balanced by the same buffer by syringeinjection. The column was then loaded on the system by the followingprocedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100 ml 10 mMTris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed by a 10 ml10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions were thentested for activity. The fractions with highest activity were furtherconcentrated by Vivaspin® 15R (Vivascience, now Sartorius VivascienceGmbH, Goettingen, Germany). The concentrated BclI-HF was then added toan equal volume of glycerol and stored at −20° C.

5. Comparison of BclI-HF and BclI-WT

The FIs of BclI-HF and BclI-WT have been determined separately on dam−lambda DNA in four NEB buffers with diluent A. The comparison is shownin FIG. 8, and the result is listed in Table 13 (below).

TABLE 13 Comparison of BclI-HF and BclI-WT BclI-HF BclI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1 12.5%  ≧250  50% 120 ≧2 NEB1 100%  ≧500 100%  32 ≧16 NEB3   25%  ≧32  50%  64 ≧½ NEB4  100% ≧2000100%  32 ≧60 BclI-HF performed best in NEB2 and NEB4, in which the bestFI was ≧2000; BclI-WT performed best in NEB2 and NEB4, in which the FIwas 32. The overall improvement factor is ≧2000/32 = ≧64.

Example 13 Engineering of HF BglII 1. Expression of BglII

BglII was expressed in E. coli transformed with pLacZZ-BglIIR andpACYC-BglIIM, each contains BglII endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of BglII-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 2, 4, 6, 7, 9, 10, 12, 13, 16, 18, 20,21, 22, 24, 25, 26, 29, 30, 33, 35, 37, 38, 39, 41, 42, 45, 48, 49, 53,54, 55, 58, 59, 60, 64, 65, 66, 67, 68, 69, 74, 75, 76, 77, 78, 81, 82,84, 85, 87, 88, 89, 90, 93, 95, 96, 97, 98, 101, 104, 105, 106, 108,109, 110, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 124, 125,131, 132, 134, 135, 136, 139, 140, 141, 142, 146, 147, 149, 150, 151,153, 154, 157, 159, 161, 162, 166, 172, 173, 174, 175, 176, 177, 179,182, 183, 184, 187, 188, 189, 191, 192, 193, 195, 196, 197, 198, 199,201, 203, 206, 207, 208, 209, 211, 212, 213, 214, 215, 216, 217, 219,222; while Tyr is changed to Phe at the positions of 8, 56, 99, 144,145, 158, 185, and 190.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER3081.

3. Selection of BglII-HF

Selection of BglII-HF was achieved using comparison of activity in NEB3and NEB4 using pXba DNA as substrate. BglII-WT has more activity inNEB3, so the mutants with more activity in NEB4 were selected. Allmutants with more activity were then compared to WT activity in glycerolto check for star activity. Normally the mutant with the highestactivity in NEB4 is the one with improved star activity. The mutantsthat were most promising (H10A, N208A, K48A, K74A, R75A, Y56F, K58A,M117A) were finally tested with ExoI buffer in water, which can promotestar activity in BglI-WT. One mutant, N208A showed decreased staractivity in NEB4 and increased overall activity. In small culture, thismutant can appear to is have stable partial activity, which we havedetermined is another indicator that the fidelity has changed.BglII(N208A) is designated as BglII-HF.

4. Purification of BglII-HF

Two liters of cell ER3081(pLacZZ-BglII(N208A), pACYC-BglIIM)) were grownin LB with 100 μg/ml Amp and 33 μg/ml at 30° C. for overnight. The cellswere harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mMNaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated BglII-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of BglII-HF and BglII-WT

The FIs of BglII-HF and BglII-WT have been determined separately on pXbaDNA in four NEB buffers with diluent B. The comparison is shown in FIG.9, and the result is listed in Table 14 (below).

TABLE 14 Comparison of BglII-HF and BglII-WT BglII-HF BglII-WTImprovement Buffer Activity FI Activity FI Factor NEB1 12.5%  ≧8000  25% 250  ≧32 NEB1  100% ≧128000  100%  64 ≧2000 NEB3   50%  ≧2000 100% 120  ≧16 NEB4   25%  ≧32000  6.3%  16 ≧2000 BglII-HF performedbest in NEB2, in which the FI was ≧128000; BglII-WT performed best inNEB3, in which the FI was 120. The overall improvement factor was≧128000/120 = ≧1000.

Example 14 Engineering of HF BstEII 1. Expression of BstEII

BstEII was expressed in E. coli transformed with pUC19-BstEIIR andpACYC-BstEIIM, each contains BstEII endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of BstEII-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 9, 10, 14, 17, 20, 21, 22, 25, 26,29, 30, 32, 36, 37, 40, 41, 44, 47, 48, 49, 50, 51, 52, 54, 57, 58, 60,61, 62, 63, 64, 65, 67, 68, 69, 72, 75, 76, 79, 80, 81, 82, 83, 85, 88,89, 90, 91, 92, 94, 95, 98, 99, 101, 102, 103, 105, 106, 111, 112, 113,116, 117, 118, 119, 120, 121, 122, 123, 130, 132, 133, 134, 135, 136,137, 138, 140, 142, 143, 147, 150, 151, 152, 154, 155, 157, 160, 161,162, 163, 165, 166, 167, 171, 172, 175, 176, 178, 179, 180, 182, 184,189, 190, 191, 192, 193, 194, 195, 199, 202, 204, 205, 206, 207, 208,209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 224,225, 227, 228, 232, 233, 234, 236, 238, 243, 244, 245, 246, 247, 251,252, 255, 256, 258, 261, 262, 264, 265, 266, 272, 274, 277, 278, 279,281; while Tyr is changed to Phe at the positions of 8, 15, 24, 27, 35,43, 77, 129, 131, 139, 156, 188, 203, 229, 257, and 263.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed is into E. colistrain ER2683.

3. Selection of BstEII-HF

Selection of BstEII-HF was achieved using comparison of activity in NEB3and NEB4 using lambda DNA as substrate. WT BstEII has more activity inNEB3, so the mutants with more activity in NEB4 were selected. Sevenmutants were found to have improved activity in NEB4: K014A, Q069A,E099A, R105A, R117A, G135A, and Y035F. R105A had the most difference inactivity compared to WT in NEB4 and water and also showed decreased staractivity when with tested in glycerol with ExoI buffer, a conditionwhich shows star activity in WT. BstEII(R105A) is designated asBstEII-HF.

4. Purification of BstEII-HF

Two liters of cell ER2683(pUC19-BstEII(R105A), pACYC-BstEIIM)) weregrown in LB with 100 μg/ml Amp and 33 μg/ml at 30° C. for overnight. Thecells were harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated BstEII-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of BstEII-HF and WT BstEII

The FIs of BstEII-HF and WT BstEII have been determined separately onlambda DNA in four NEB buffers with diluent A. The is comparison isshown in FIG. 10, and the result is listed in Table 15 (below).

TABLE 15 Comparison of BstEII-HF and BstEII-WT BstEII-HF BstEII-WTImprovement Buffer Activity FI Activity FI Factor NEB1   3%  ≧64  50% 16 ≧4 NEB1   50% ≧1000 100%  4 ≧250 NEB3  1.6%  ≧32  50% 16  ≧2 NEB4  100%≧2000 100%  4 ≧250 BstEII-HF performed best in NEB4, in which the FI was≧2000; BstEII-WT performed best in NEB2 and NEB4, in which the FI was 4.The overall improvement factor is ≧2000/4 = ≧500.

Example 15 Engineering of HF BanII 1. Expression of BanII

BanII was expressed in E. coli transformed with pUC19-BanIIR andpACYC1-BanIIM, each contains BanII endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of BanII-HF

All residues except Tyr (and those that were already Ala) were changedto Ala at positions 7, 8, 9, 10, 12, 16, 17, 20, 21, 23, 24, 25, 26, 28,29, 24, 31, 32, 35, 38, 39, 43, 44, 45, 47, 49, 54, 59, 61, 63, 64, 66,67, 71, 72, 73, 74, 75, 77, 78, 81, 83, 84, 87, 88, 92, 94, 95, 96, 97,99, 100, 103, 104, 105, 106, 107, 108, 111, 112, 113, 115, 117, 118,120, 121, 122, 123, 126, 127, 128, 129, 130, 131, 135, 139, 142, 143,145, 146, 147, 148, 149, 152, 153, 155, 156, 163, 166, 167, 168, 169,170, 171, 173, 175, 176, 178, 179, 180, 181, 183, 184, 186, 190, 191,194, 195, 196, 198, 199, 200, 207, 208, 211, 213, 214, 215, 216, 219,220, 221, 222, 224, 226, 229, 230, 231, 232, 234, 235, 236, 237, 239,240, 242, 245, 246, 247, 248, 252, 254, 256, 257, 258, 259, 261, 262,263, 264, 266, 267, 270, 271, 272, 274, 276, 278, 279, 281, 284, 285,286, 287, 289, 291, 292, 293, 294, 295, 296, 300, 302, 303, 305, 309,311, 312, 314, 317, 318, 319, 322, 326, 327, 328, 330, 331, 334, 338,339, 341, 342, 344, 346, 347, 348, 349, 351, 352, 355, 356, and 358; Tyrwas changed to Phe at the positions of 27, 50, 80, 160, 182, 197, 244,251, 260, 307, and 313.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2566.

3. Selection of BanII-HF

Selection of BanII-HF was achieved using by comparing the of activity inNEB4 with water with the star activity in ExoI buffer and glycerol,using lambda DNA as substrate. Mutants which showed similar or improvedactivity to WT in water and NEB4, while also showing improved staractivity were selected for further testing. These mutants include N106A,Q169A, and E314A. R126A was also chosen because it showed a consistentpartial pattern, which we have also shown to be an indicator of highfidelity. After purification, R126A showed the best decrease in staractivity. BanII(R126A) is designated BanII-HF.

4. Purification of BanII-HF

Two liters of cell ER2566(pUC19-BanII(R126A), pACYC-BanIIM)) were grownin LB with 100 μg/ml Amp and 33 μg/ml at 30° C. for overnight. The cellswere harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mMNaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated BanII-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of BanII-HF and BanII-WT

The FIs of BanII-HF and BanII-WT have been determined separately on dam−lambda DNA in four NEB buffers with diluent A. The result is listed inTable 16 (below).

TABLE 16 Comparison of BanII-HF and BanII-WT BanII-HF BanII-WTImprovement Buffer Activity FI Activity FI Factor NEB1  100% ≧4000  100%64  ≧64 NEB1   50% ≧2000  100% 64  ≧32 NEB3 12.5%  ≧500 12.5% 16  ≧32NEB4   50% ≧2000  100% 16 ≧125 BanII-HF performed best in NEB1, in whichthe FI was ≧4000; BanII-WT performed best in NEB1, NEB2 and NEB4, inwhich the best FI was 64. So the overall improvement factor in NEB1 is≧4000/64 = ≧64.

Example 16 Engineering of HF PspGI 1. Expression of PspGI

PspGI was expressed in E. coli transformed with pRRS-PspGIRM whichcontains PspGI endonuclease and methylase gene. The cells were grown at30° C. overnight in LB with Amp.

2. Mutagenesis of PspGI-HF

The length of PspGI protein is 272 amino acids. Total 166 AA sites ofPspGI protein were initially designed to be mutated into Ala (or Phe).Cys, Asp, Glu, Phe, His, Lys, Met, Asn, Gln, Arg, Ser, Thr, Trp were ismutated to Ala. Try was mutated to Phe. These were: 8, 10, 11, 12, 13,14, 15, 16, 18, 19, 20, 21, 22, 25, 26, 29, 30, 32, 34, 35, 38, 39, 42,43, 44, 45, 46, 47, 48, 51, 52, 53, 54, 57, 60, 61, 62, 65, 68, 69, 71,72, 73, 75, 76, 80, 82, 84, 85, 86, 87, 89, 90, 91, 93, 94, 96, 98, 99,100, 101, 102, 105, 109, 110, 113, 134, 135, 136, 137, 138, 142, 143,145, 149, 150, 151, 152, 153, 158, 160, 161, 162, 164, and 165. Themutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain 2984.

3. Selection of PspGI-HF

Selection of PspGI-HF was achieved using comparison of mutants and WT'sactivity in NEB4 using pBC4 DNA as substrate. The selection assays ofPspGI were performed using pBC4 as substrate in NEB4 (2 h digestion at69° C.). 11 mutants are found to have more activity in NEB4 than WT:T20A, P52A, Y67F, K68A, R75A, E86A, Q90A, S91A, Q93A, H121A and G172A.PspGI (R75A) has much higher activity than WT in NEB4. Normally the onewith highest activity in NEB4 is the one with improved star activity.After several rounds of comparison in different conditions andsubstrates, PspGI (R75A) was found to be the preferred mutant, retaininghigh cleavage high activity, but displaying substantially reduced staractivity. PspGI (R75A) is designated as PspGI-HF.

4. Purification of PspGI-HF

Two liters of cell E. coli 2984 (pRRS-PspGIRM (R75A)) were grown in LBwith 100 μg/ml Amp at 30° C. for overnight. The cells were harvested andsonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl. After acentrifugation at 15,000 rpm for 30 minutes, the supernatant was loadedon the 5 ml HiTrap™ Heparin HP column (GE Healthcare, now Pfizer, Inc.,Piscataway, N.J.) pre-balanced by the same buffer by syringe injection.The column was then loaded on the system by the following procedure: 48ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100 ml 10 mM Tris-HCl, pH 7.5, 50mM-1M NaCl linear gradient and followed by a 10 ml 10 mM Tris-HCl, pH7.5, 1M NaCl step. The eluted fractions are then test for activity. Thefractions with highest activity were further concentrated. Theconcentrated PspGI-HF was then added same volume of glycerol and storedin the −20° C. condition.

5. Comparison of PspG-HF and PspGI-WT

The FIs of PspG-HF and PspGI-WT have been determined separately on pBC4DNA in four NEB buffers with diluent A. The result is listed in Table 17(below).

TABLE 17 Comparison of PspG-HF and PspGI-WT PspGI-HF PspGI-WTImprovement Buffer Activity FI Activity FI Factor NEB1  25% ≧1000 12.5%1 ≧1000 NEB1 100% ≧4000  100% 4 ≧1000 NEB3 100% ≧4000  100% 8  ≧500 NEB4100% ≧4000  100% 1 ≧4000 PspGI-HF performed best in at NEB2, NEB3 andNEB4, in which the preferred FI was ≧4000; PspGI-WT performed best inNEB2, NEB3 and NEB4. The preferred FI of PspGI-WT in NEB3 was 8. Theoverall FI improvement factor was ≧4000/8 = ≧500.

Example 17 Engineering of HF SpeI 1. Expression of SpeI

SpeI was expressed in E. coli transformed with pRRS-SpeI andpASYX20-SpeIM9, each contains SpeI endonuclease and methylase gene. Thecells were grown at 30° C. overnight in LB with Amp and Kan.

2. Mutagenesis of SpeI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 9, 10, 17, 18, 20, 21, 22, 24, 25,26, 29, 30, 31, 32, 33, 34, 36, 40, 43, 45, 46, 49, 50, 51, 52, 53, 54,57, 58, 59, 61, 65, 66, 70, 73, 74, 75, 76, 77, 78, 80, 81, 84, 86, 87,88, 89, 90, 92, 96, 97, 101, 102, 103, 105, 107, 108, 109, 110, 112,113, 115, 116, 118, 121, 122, 125, 126, 128, 130, 131, 137, 138, 139,140, 142, 146, 149, 151, 152, 154, 157, 158, 159, 160, 161, 163, 166,167, 169, 170, 172, 174, 175, 179, 180, and 182; Tyr was changed to Pheat the positions of 13, 19, 28, 55, 104, 120, 129, and 164.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER1038.

3. Selection of SpeI-HF

Selection of SpeI-HF was achieved using by comparing the activity ofeach mutant in NEB4 with water and pXBA DNA that was previously digestedwith SacI-HF as substrate, to a glycerol reaction with ExoI and normalpXba. The SacI-HF digested pXBA allowed for greater clarity when testingmutants for activity compared to WT. The glycerol reaction was is usedto compare star activity results. Several mutants showed high cognateactivity with a simultaneous decrease in star activity: E059A, P065A,S108A, N172A, K174A, Q179A, G182A, and Y055F. After comparing purifiedsamples, SpeI(P065A) was designated as SpeI-HF.

4. Purification of SpeI-HF

Two liters of cell ER3081(pRRS-SpeIM7(P065A), pSYX20-SpeIM9)) were grownin LB with 100 μg/ml Amp and 33 μg/ml at 30° C. for overnight. The cellswere harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mMNaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated SpeI-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of SpeI-HF and SpeI-WT

The FIs of SpeI-HF and SpeI-WT have been determined separately on pXbaDNA in four NEB buffers with diluent C. and the result is listed inTable 18 (below).

TABLE 18 Comparison of SpeI-HF and SpeI-WT SpeI-HF SpeI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1   50% ≧4000  100% 1000 ≧1000NEB1 12.5% ≧2000   50%  500   ≧2 NEB3 12.5% ≧2000 12.5% 2000   ≧1/8 NEB4 100% ≧8000   50%  500   ≧2 SpeI-HF has most activity in NEB4, where theFI is ≧8000; SpeI-WT has most activity in NEB1, where the FI is 1000. Sothe overall improvement factor is ≧8.

Example 18 Engineering of HF BsmAI 1. Expression of BsmAI

BsmAI was expressed in E. coli transformed with pBAD241-BsmAIR andpACYC-BsmAIM, each contains BsmAI endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Cam and theninduced by arabinose for 4 hours.

2. Mutagenesis of BsmAI-HF

Due to the homology among BsaI, BsmBI and BsmAI, amino acids in theregion 210-227 of BsmAI were selected to mutate to Ala one at a timebecause that the high fidelity mutants of BsaI and BsmBI were found inthe this similar region.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER3081.

3. Selection of BsmAI-HF

Selection of BsmAI-HF was achieved using comparison of star activity ofmutant BsmAI and WT BsmAI in NEB4 on FX174 DNA as substrate. Two mutantshad less star activity than the WT BsmAI: N212A and L213A. MutantBsmAI(N212A) is designated as BsmAI-HF.

4. Purification of BsmAI-HF

Two liters of cell ER2566(pBAD241-BsmAI(N212A), pACYC184-BsmAIM)) weregrown in LB with 100 μg/ml Amp and 33 μg/ml Cam at 37° C. for overnight.Then the cells were induced by arabinose with final concentration of0.2% for 4 hours. The cells were harvested and sonicated in 20 ml 10 mMTris-HCl, pH 7.5, 50 mM NaCl. After a centrifugation at 15,000 rpm for30 minutes, the supernatant was loaded on the 5 ml HiTrap™ Heparin HPcolumn (GE Healthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balancedby the same buffer by syringe injection. The column was then loaded onthe system by the following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl, 100 ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradientand followed by a 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The elutedfractions are then test for activity. The fractions with highestactivity were further concentrated by Vivaspin® 15R (Vivascience, nowSartorius Vivascience GmbH, Goettingen, Germany). The concentratedBsmAI-HF was then added same volume of glycerol and stored at −20° C.

5. Comparison of BsmAI-HF and BsmAI-WT

The FIs of BsmAI-HF and BsmAI-WT have been determined separately onFX174 DNA in four NEB buffers with diluent B. The result is listed inTable 19 (below).

TABLE 19 Comparison of BsmAI-HF and BsmAI-WT BsmAI-HF BsmAI-WTImprovement Buffer Activity FI Activity FI Factor NEB1  100% ≧4000  50%120 ≧32 NEB1   50% ≧2000  50% 500  ≧4 NEB3 12.5%  ≧500  50% 500  1 NEB4 100% ≧4000 100% 250  ≧8 BsmAI-HF performed best in NEB1 and NEB4, inwhich the FI was ≧4000; BsmAI-WT performed best in NEB4, in which the FIwas 250. So the overall improvement factor was ≧4000/250 = ≧16.

Example 19 Engineering of HF BstXI

BstXI recognizes and digests at CCANNNNN/NTGG as described in Example 19of International Publication No. WO 2009/009797. A mutant BstXI(N65A)was selected as the high fidelity version of the BstXI. A further stepto search for better BstXI with less star activity is to mutate N65 toall other amino acid residues. Among those, BstXI(N65T) was found tohave less star activity and designated to be BstXI-HF.

The BstXI-HF was expressed in ER2833 (pBAD241-BstXI(N65T), pACYC-BstXIM.The growth and purification methods were performed according toWO/2009/009797.

The following table (Table 20) compares the FIs of BstXI-HF and BstXIWT.

TABLE 20 Comparison of BstXI-HF and BstXI-WT BstXI-HF BstXI-WTImprovement Buffer Activity FI Activity FI Factor NEB1  50% ≧500  6% 4≧125 NEB2 100% ≧1000 100% 32 ≧32 NEB3 100% ≧1000 100% 2 ≧500 NEB4 100%≧1000 100% 32 ≧32 The BstXI-HF had the best activity in NEB2, NEB3 andNEB4, the best FI of BstXI-HF was ≧1000; the WT BstXI had the bestactivity in NEB2, NEB3 and NEB4. The FI of WT BstXI in NEB2 and NEB4 was32. So the overall improvement factor was ≧32.

Example 20 Engineering of HF SfiI 1. Expression of SfiI

SfiI was expressed in E. coli transformed with pRRS-SfiIR andpSX33-SfiIM, each contains SfiI endonuclease and methylase gene. Thecells were grown at 30° C. overnight in LB with Amp and Kan.

2. Mutagenesis of SfiI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 9, 11, 12, 14, 15, 17, 18, 19, 20,22, 23, 26, 29, 30, 32, 33, 34, 36, 37, 40, 41, 42, 45, 46, 47, 48, 49,55, 56, 58, 59, 63, 66, 67, 69, 71, 72, 73, 76, 79, 81, 82, 84, 87, 88,89, 90, 91, 94, 95, 100, 102, 104, 105, 106, 107, 108, 109, 110, 111,113, 114, 115, 116, 118, 120, 122, 124, 125, 126, 127, 128, 129, 130,133, 135, 137, 140, 141, 145, 146, 148, 149, 150, 153, 156, 157, 158,162, 166, 167, 169, 170, 172, 173, 174, 176, 177, 179, 180, 185, 187,188, 190, 192, 193, 194, 196, 197, 198, 199, 200, 201, 202, 205, 207,208, 209, 210, 211, 213, 214, 215, 218, 220, 224, 225, 227, 228, 231,233, 235, 236, 238, 240, 242, 243, 244, 246, 247, 248, 249, 251, 252,254, 255, 257, 258, 259, 261, 262, 263; Tyr is changed to Phe at thepositions of 31, 60, 68, 80, 164, 165, 175, 182, 195, 222, 239, and 245.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2169.

3. Selection of SfiI-HF

Selection of SfiI-HF was achieved using comparison of activity betweenmutants and WT in water with NEB ExoI buffer and BSA using pXba DNApredigested with EcoRI-HF as substrate. Mutants with similar or greateractivity to wild type while also showing a change in star activity in adefined buffer compared to WT were selected. Several mutants are foundto have more activity in NEB4: E007A, D011A, E049A, R073A, R0114A,G137A, S210A, and R213A. After purification, P114A proved to have themost significant decrease in star activity. SfiI(R114A) is designated asSfiI-HF.

Also notable were the mutants that increased star activity: N071A,D079A, H162A, R225A, K227A, Y068F, and Y182F. Y068F was previously notedto have different cleavage from WT.

4. Purification of SfiI-HF

Two liters of cell ER2169(pRRS-SfiI(R114A), pSX33-SfiIM)) were grown inLB with 100 μg/ml Amp and 33 μg/ml Kan at 30° C. for overnight. Thecells were harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated SfiI-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of SfiI-HF and SfiI-WT

The FIs of SfiI-HF and SfiI-WT have been determined separately on pBC4DNA in four NEB buffers with diluent C. The comparison is shown in FIG.11, and the result is listed in Table 21 (below).

TABLE 21 Comparison of SfiI-HF and SfiI-HF SfiI-HF SfiI-HF ImprovementBuffer Activity FI Activity FI Factor NEB1   50% ≧250 12.5% 64 ≧4 NEB212.5% ≧1000  100% 250 ≧4 NEB3  0.4% ≧32  100% 2000 ≧ 1/64 NEB4  100%≧8000   25% 64 ≧125 SfiI-HF performed best in NEB4, in which the FI was≧8000; WT SfiI performed best in NEB3, in which the FI was 2000. Theoverall improvement factor is ≧8000/2000 = ≧4.

Example 21 Engineering of HF PmeI 1. Expression of PmeI

PmeI was expressed in E. coli transformed with pRRS-PmeIR andpACYC184-EsaS9IM, each contains PmeI endonuclease and methylase gene.The cells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of PmeI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 8, 10, 13, 14, 17, 20, 21, 22, 25,28, 29, 30, 32, 33, 35, 37, 39, 41, 42, 43, 46, 47, 49, 50, 51, 54, 55,60, 62, 63, 64, 66, 67, 68, 69, 71, 72, 73, 77, 79, 80, 81, 82, 83, 86,87, 91, 94, 95, 96, 97, 98, 100, 104, 106, 107, 108, 109, 110, 112, 113,114, 115, 116, 117, 118, 121, 123, 124, 127, 130, 131, 132, 133, 134,135, 137, 138, 145, 147, 148, 149, 151, 152, 153, 154, 155, 157, 160,162, 165, 166, 167, 169, 170, 171, 172, 177, 180, 181, 182, 183, 185,186, 188, 190, 191, 192, 193, 194, 199, 200, 201, 202, 204, 207, 208,209, 210, 211, 212, 215, 218, 219, 221, 222, 223, 225; Tyr is changed toPhe at the positions of 111, 129, 146, and 161.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2426.

3. Selection of PmeI-HF

Selection of PmeI-HF was achieved using comparison of activity betweenWT and mutants in water NEB4 using lambda DNA as substrate with the samemutants in glycerol with NEB Thermopol buffer and pXba as a substrate.The testing of mutants and WT PmeI in water on lambda DNA allowed for areference of cognate activity, and with similar or more activity than WTin NEB4 were selected. Mutants with acceptable activity were thenrejected if they showed no change in star activity when tested underglycerol conditions with Thermopol buffer and pXba. Several mutants wereshown to have differences in star activity: P079A, E086A, H096A, andE218A. PmeI(E086A) is designated as PmeI-HF.

4. Purification of PmeI-HF

Two liters of cell ER2426(pRRS-PmeI(P154A), pACYC184-EsaS9IM)) weregrown in LB with 100 μg/ml Amp and 33 μg/ml Cam at 37° C. overnight. Thecells were harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated PmeI-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of PmeI-HF and PmeI-WT

The FIs of PmeI-HF and PmeI-WT have been determined separately on pXbaDNA in four NEB buffers with diluent A. The result is listed in Table 22(below).

TABLE 22 Comparison of PmeI-HF and PmeI-WT PmeI-HF PmeI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1 12.5% ≧2000 100% 250 ≧64 NEB2 6.3% ≧500 100% 250 ≧500 NEB3  0.4% ≧32  50% 120 ≧125 NEB4  100% ≧8000 25%  64 ≧500 PmeI-HF performed best in NEB4, in which the FI was ≧8000;PmeI-WT performed best in NEB1 and NEB2, in which the FI was 250. Theoverall improvement factor is ≧8000/250 = ≧16.

Example 22 Engineering of HF SmaI 1. Expression of SmaI

SmaI was expressed in E. coli transformed with pRRS-SmaIR andpSYX20-SmaIM, each contains SmaI endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Kan.

2. Mutagenesis of SmaI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala; all Tyr were changed to Phe.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2428.

3. Selection of SmaI-HF

Selection of SmaI-HF was achieved using comparison of activity in waterNEB4 using pXba DNA as substrate with a star-activity producing glycerolcondition with NEB Standard Taq buffer. Mutants which showed changes instar activity in the designated buffer while retaining similar or highcognate activity to WT were selected. Several mutants were found: E32R,S081A, G132A and a double-mutant F60L/S61R. SmaI(F60L/S61R) isdesignated as SmaI-HF.

4. Purification of SmaI-HF

Two liters of cell ER2428(pRRS-SmaI(F60L/S61R), pSYX20-SmaIM)) weregrown in LB with 100 μg/ml Amp and 33 μg/ml Kan at 37° C. overnight. Thecells were harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated SmaI-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of SmaI-HF and SmaI-WT

The FIs of SmaI-HF and WT SmaI have been determined separately on pXbaDNA in four NEB buffers with diluent A. The comparison is shown in FIG.12, and the result is listed in Table 23 (below).

TABLE 23 Comparison of SmaI-HF and SmaI-WT SmaI-HF SmaI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1   0.2% ≧2000   3% ≧16 ND NEB2  3.2% ≧32000 12.5% ≧64 ND NEB3 0.0032% ≧32  0.8% ≧8 ND NEB4   100%≧256000  100%  64 ≧4000 ND: Not determinable SmaI-HF performed best inNEB4, in which the FI was ≧256000; SmaI-WT performed best in NEB2 andNEB4, in which the FI was 64. The overall improvement factor is≧256000/64 = ≧4000.

Example 23 Engineering of High Fidelity AatII 1. Expression of AatII

AatII was expressed in E. coli transformed with pRRS-AatIIR andpACYC184-AatIIM, each contains AatII endonuclease and methylase gene.The cells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of AatII-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 8, 9, 11, 12, 13, 16, 17, 18, 20, 22,26, 29, 32, 33, 35, 36, 37, 38, 40, 43, 45, 46, 49, 52, 53, 54, 56, 57,58, 60, 61, 62, 64, 65, 69, 70, 71, 72, 73, 74, 75, 77, 79, 80, 83, 84,86, 87, 90, 92, 93, 94, 95, 97, 99, 100, 103, 104, 106, 107, 111, 113,114, 117, 121, 123, 124, 125, 126, 128, 129, 131, 132, 133, 135, 136,140, 141, 143, 144, 145, 146, 148, 149, 150, 151, 153, 155, 156, 157,160, 164, 165, 167, 169, 171, 172, 173, 174, 175, 176, 177, 179, 181,182, 186, 189, 191, 192, 193, 194, 196, 198, 200, 201, 203, 204, 205,206, 207, 208, 210, 211, 213, 214, 216, 217, 219, 220, 221, 222, 226,228, 230, 231, 233, 235, 236, 237, 238, 240, 241, 244, 247, 248, 249,250, 251, 252, 253, 256, 262, 264, 265, 266, 268, 269, 272, 273, 275,280, 281, 282, 283, 286, 298, 292, 293, 295, 296, 297, 298, 301, 302,308, 309, 311, 312, 313, 314, 315, 317, 319, 321, 325, 327, 329, 330,333, 334, 335, 336; Tyr was changed to Phe at the positions of 82, 89,98, 112, 232, 305, and 306.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2426.

3. Selection of AatII-HF

Selection of AatII-HF was achieved using comparison of activity in NEB4in water to NEB ExoI buffer in glycerol using pXba DNA as substrate.Mutants which showed changes in star activity under the glycerolconditions were chosen for further testing as long as they had similaror greater activity than WT under normal conditions in water. Severalmutants were chosen for further testing after the initial screen: G013A,G016A, K018A, P052A, R053A, K070A, E071A, D072A, G073A, S84A, E086A,R090A, K094A, R095A, P099A, P103A, K113A, N135A, S151A, P157A, G173A,T204A, 5206A, K207A, E233A, N235A, E237A, S238A, D241A, K295A, S301A,and 5302A. AatII(N235A) is designated as AatII-HF.

4. Purification of AatII-HF

Two liters of cell ER2426(pRRS-AatII(N235A), pACYC184-AatIIM)) weregrown in LB with 100 μg/ml Amp and 33 μg/ml Cam at 37° C. overnight. Thecells were harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated AatII-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of AatII-HF and AatII-WT

The FIs of AatII-HF and WT AatII have been determined separately onpBR322 DNA in four NEB buffers with diluent A. The result is listed inTable 24 (below).

TABLE 24 Comparison of AatII-HF and AatII-WT Improve- AatII-HF AatII-WTment Buffer Activity FI Activity FI Factor NEB1 NC NC  3% 32 ND NEB2 NCNC 100% ¼ ND NEB3 NC NC NC NC ND NEB4 100% ≧1000  50% 16 ≧64 NC: Notcompletable; ND: Not determinable AatII-HF performed best in NEB4, inwhich the FI was ≧1000; WT AatII performed best in NEB2, in which the FIwas ¼. The overall improvement factor is ≧1000/¼ = ≧4000.

Example 24 Engineering of HF ApoI 1. Expression of ApoI

ApoI was expressed in E. coli transformed with pRRS-ApoIR andpACYC184-ApoIM, each contains ApoI endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of ApoI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, and Arg, werechanged to Ala at positions 8, 9, 10, 11, 13, 14, 17, 18, 19, 20, 21,22, 23, 24, 26, 28, 29, 33, 35, 36, 37, 39, 41, 43, 47, 48, 49, 50, 51,56, 57, 60, 62, 63, 64, 66, 67, 69, 71, 72, 73, 75, 76, 77, 80, 81, 82,83, 84, 87, 92, 93, 94, 95, 96, 97, 102, 103, 105, 106, 107, 108, 109,110, 111, 113, 115, 116, 117, 119, 120, 121, 124, 125, 128, 129, 131,132, 133, 136, 137, 143, 144, 145, 148, 153, 155, 157, 159, 160, 161,162, 163, 166, 167, 169, 170, 175, 176, 178, 179, 181, 184, 185, 186,187, 188, 189, 192, 193, 194, 195, 199, 201, 202, 204, 206, 207, 209,210, 214, 216, 217, 218, 221, 226, 227, 229, and 230.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2426.

3. Selection of ApoI-HF

Selection of ApoI-HF was achieved using comparison of activity in NEB3and NEB4 using lambda DNA as substrate. Mutants with more activity thanWT in NEB4 were selected as increased activity in NEB4 is an indicatorof improved fidelity. The following mutants are found to have moreactivity in NEB4: S64A, S80A, S162A, T77A/T96A and N178A.ApoI(T77A/T96A) is designated as ApoI-HF.

4. Purification of ApoI-HF

Two liters of cell ER2426(pRRS-ApoI(T77A/T96A), pACYC184-ApoIM)) weregrown in LB with 100 μg/ml Amp and 33 μg/ml Cam at 37° C. overnight,induced with 0.5 mM ITPG after 8 hours of growth. The cells wereharvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl.After a centrifugation at 15,000 rpm for 30 minutes, the supernatant wasloaded on the 5 ml HiTrap™ Heparin HP column (GE Healthcare, now Pfizer,Inc., Piscataway, N.J.) pre-balanced by the same buffer by syringeinjection. The column was then loaded on the system by the followingprocedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100 ml 10 mMTris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed by a 10 ml10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions were thentested for activity. The fractions with highest activity were furtherconcentrated by Vivaspin® 15R (Vivascience, now Sartorius VivascienceGmbH, Goettingen, Germany). The concentrated ApoI-HF was then added anequal volume of glycerol and stored at −20° C.

5. Comparison of ApoI-HF and ApoI-WT

The FIs of ApoI-HF and ApoI-WT have been determined separately on pXbaDNA in four NEB buffers with diluent A. The result is listed in Table 24(below).

TABLE 24 Comparison of ApoI-HF and ApoI-WT ApoI-HF ApoI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1  50% ≧2000  25% 120 ≧16 NEB2100% ≧4000 100%  32 ≧125 NEB3  25% ≧1000 100%  64 ≧16 NEB4  50% ≧2000 50%  32 ≧64 ApoI-HF performed best in NEB2, in which the FI was ≧4000;WT Apol performed best in NEB2 and NEB3, in which the best FI was 64.The overall improvement factor is ≧4000/64 = ≧64.

Example 25 Engineering of High Fidelity BsmBI

BsmBI recognizes and digests at CGTCTCN1/N5 as described in Example 23of International Publication No. WO 2009/009797. A mutant BsmBI(R232A)was selected as the high fidelity version of the BsmBI. Furthercharacterization of this mutant revealed that though the performance ofBsmBI(R232A) on one hour scale is excellent, it did not perform well inthe overnight digestion. While searching for more mutants, BsmBI(W238A)was found to be excellent in both one hour and overnight reaction, anddesignated to be BsmBI-HF (FIG. 13).

The BsmBI-HF was expressed in ER3081(pBAD241-BsmBIR(W238A)/pACYC-BsmAIM). The growth and purificationmethods were performed according to WO/2009/009797.

The following table (Table 26) compares the FIs of BsmBI-HF andBsmBI-WT.

TABLE 26 Comparison of BsmBI-HF and BsmBI-WT Improve- BsmBI-HF BsmBI-WTment Buffer Activity FI Activity FI Factor NEB1   50%  32 12.5% 1 32NEB2   50% 120   50% 8 25 NEB3 12.5% 250  100% 120  2 NEB4  100% 250  25% 4 64 The BsmBI-HF had the best activity in NEB4, the FI ofBsmBI-HF in NEB4 was 250; the BsmBI-WT had the best activity in NEB3.The FI of WT BsmBI in NEB2 was 120. So the overall improvement factorwas 2.

Example 26 Engineering of HF BmtI 1. Expression of BmtI

BmtI was expressed in E. coli transformed with pACYC-BmtIM andplaczz1-BmtIR. pACYC is a low copy compatible plasmid. The cells weregrown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of BmtI-HF

The point mutagenesis of the selected mutations was done by inverse PCR.150 amino acid mutations were made in BmtI as follows. Cys, Asp, Glu,Phe, His, Lys, Met, Asn, Gln, Arg, Ser, Thr, Trp were mutated to Ala.Try was mutated to Phe. These were: 5, 9, 11, 12, 16, 19, 20, 23, 24,25, 26, 27, 30, 32, 33, 34, 35, 36, 39, 45, 46, 49, 50, 51, 53, 56, 58,59, 60, 63, 65, 69, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 83, 85, 86,88, 89, 90, 92, 93, 94, 95, 97, 98, 99, 101, 104, 105, 106, 108, 110,111, 112, 113, 116, 118, 119, 120, 121, 122, 124, 128, 129, 131, 132,133, 134, 136, 138, 139, 140, 141, 142, 144, 145, 146, 147, 148, 150,151, 152, 154, 156, 157, 161, 162, 163, 165, 166, 167, 168, 169, 171,172, 173, 175, 178, 179, 180, 181, 185, 186, 189, 190, 191, 193, 194,195, 196, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211,213, 214, 216, 217, 218, 219, 220, 221, 222, 226, 228, 229, 230, 231,234, 236, 237, 238, 239 and 241. The mutagenesis methods were inversePCR with paired primers followed by DpnI digestion. The treated productwas then transformed into E. coli strain 3081.

3. Selection of BmtI-HF

Four colonies of each mutation were grown up in LB with Amp and Cam at37° C. overnight. The standard cognate and star activity assays of BmtIwere performed using pBC4 in ExoI buffer and 10% DMSO.

The mutants S50A, Y81F, N93A and W207A were picked out in screeningassays. After several rounds of comparison in different conditions andsubstrates, S50A was found to be the preferred mutant, retaining highcanonical enzyme activity, but displaying substantially reduced staractivity. BmtI(S50A) was labeled as BmtI-HF.

4. Purification of BmtI-HF

Two liters of cell E. coli 3081 (placzz1-BmtIR(S50A), pACYC-BmtIM) weregrown in LB with 100 μg/ml Amp and 30 μg/ml Cam at 37° C. for overnight.The cells were harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5,50 mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions arethen test for activity. The fractions with highest activity were furtherconcentrated. The concentrated BmtI-HF was then added same volume ofglycerol and stored at −20° C.

5. Comparison of BmtI-HF and BmtI-WT

BmtI-HF was 2-fold serial diluted with A and reacted on pXba. The resultis shown in Table 27.

TABLE 27 Comparison of BmtI-HF and BmtI-WT BmtI-HF BmtI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1   25% ≧256000   50% 32 ≧8000NEB2   25% ≧256000  100% 16 ≧16000 NEB3  0.2% ≧2000  6.3% 32 ≧64 NEB4 100% ≧1000000  100% 16 ≧62500 BmtI-HF performed best in NEB4, in whichthe preferred FI was ≧1000000; BmtI-WT performed best in NEB2 and NEB4,where the FI was 16. The overall FI improvement factor was ≧1000000/16 =≧62500

Example 27 Engineering of HF BstNI 1. Expression of BstNI

BstNI was expressed in E. coli transformed with pBAD241-BstNIR andpACYC184-BstNIM, each contains BstNI endonuclease and methylase gene.The cells were grown at 37° C. overnight in LB with Amp and Cam, dilutedto 1/10 with LB and then induced by arabinose for 4 hours.

2. Mutagenesis and Selection of BstNI-HF

During the experiment of creating a series mutations of BstNI,BstNI(G26N) was found to have less star activity than the WT BstNI. Tosearching for better BstNI mutants with even less star activity, G26 wasis mutated to all other amino acids. Among all these mutants,BstNI(G26T) has the least star activity and is designated as BstNI-HF.

3. Purification of BstNI-HF

Two liters of cell ER2833(pBAD241-BstNI(G26T), pACYC184-BstNIM) weregrown in LB with 100 μg/ml Amp and 33 μg/ml Cam at 37° C. for overnight.Then the cells were diluted 1 to 10 with LB and then induced byarabinose with final concentration of 0.2% for 4 hours. The cells wereharvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl.After a centrifugation at 15,000 rpm for 30 minutes, the supernatant wasloaded on the 5 ml HiTrap™ Heparin HP column (GE Healthcare, now Pfizer,Inc., Piscataway, N.J.) pre-balanced by the same buffer by syringeinjection. The column was then loaded on the system by the followingprocedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100 ml 10 mMTris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed by a 10 ml10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions are then testfor activity. The fractions with highest activity were furtherconcentrated by Vivaspin® 15R (Vivascience, now Sartorius VivascienceGmbH, Goettingen, Germany). The concentrated BstNI-HF was then addedsame volume of glycerol and stored at −20° C.

4. Comparison of BstNI-HF and WT BstNI

The FIs of BstNI-HF and WT BstNI have been determined separately onpBR322 DNA in four NEB buffers with diluent A. The comparison is shownin FIG. 14, and the result is listed in Table 28 (below).

TABLE 28 Comparison of BstNI-HF and BstNI-WT BstNI-HF BstNI-WTImprovement Buffer Activity FI Activity FI Factor NEB1 50% ≧120  50% 8≧16 NEB2 100% ≧500 100% 64  8 NEB3 25% ≧120 100% 250 ≧⅛ NEB4 100%  500 50% 4 ≧32 BstNI-HF performed best in NEB2 and NEB4, in which the bestFI was ≧500; BstNI-WT performed best in NEB2 and NEB3, in which thebestFI was 250. So the overall improvement factor was ≧500/250 = ≧2.

Example 28 Engineering of HF MluI 1. Expression of MluI

MluI was expressed in E. coli transformed with pUC19-MluIR andpACYC184-MluIM, each contains MluI endonuclease and methylase gene. Thecells were grown at 30° C. overnight in LB with Amp and Cam.

2. Mutagenesis of MluI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 8, 10, 11, 13, 16, 21, 23, 24, 26,27, 30, 31, 33, 34, 35, 36, 37, 39, 42, 44, 48, 50, 51, 54, 57, 59, 60,61, 67, 68, 71, 72, 74, 75, 78, 79, 81, 83, 84, 85, 86, 89, 90, 93, 94,95, 97, 99, 101, 102, 104, 106, 108, 111, 112, 114, 116, 117, 119, 120,121, 123, 125, 128, 130, 131, 132, 134, 136, 137, 139, 140, 141, 142,144, 145, 146, 148, 152, 154, 155, 156, 157, 159, 161, 163, 165, 166,170, 172, 173, 174, 176, 177, 179, 180, 181, 182, 183, 184, 186, 189,192, 195, 196, 197, 200, 206, 207, 208, 210, 211, 214, 216, 218, 219,220, 221, 223, 227, 228, 230, 232, 233, 234, 236, 237, 238, 240, 243,244, 247, 249, 255, 256, 257, 258, 261, 263, 264, 265, 266, 269; Tyr waschanged to Phe at the positions of 14, 28, 47, 53, 77, 107, 175, 198,217, 239, and 248.

The mutagenesis methods were inverse PCR with paired primers is followedby DpnI digestion. The treated product was then transformed into E. colistrain ER1582.

3. Selection of MluI-HF

Selection of MluI-HF was achieved using comparison of activity in NEB3and NEB4 using lambda DNA as substrate. Mutants with more activity thanWT in NEB4 were selected as increased activity in NEB4 is an indicatorof improved fidelity. The only mutant found to fit our criteria wasE112A/R132A; MluI(E112A/R132A) is designated as MluI-HF.

4. Purification of MluI-HF

Two liters of cell ER1582(pUC19-MluI(E112A/R132A), pACYC184-MluIM)) weregrown in LB with 100 μg/ml Amp and 33 μg/ml Cam at 30° C. overnight. Thecells were harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated MluI-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of MluI-HF and MluI-WT

The FIs of MluI-HF and WT MluI have been determined separately on lambdaDNA in four NEB buffers with diluent A. The comparison is shown in FIG.15, and the result is listed in Table 29 (below).

TABLE 29 Comparison of MluI-HF and MluI-WT MluI-HF MluI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1   50% ≧16000   25% 500 ≧32NEB2  100% ≧32000  6.3% 16 ≧200 NEB3  6.3% ≧2000  100% 2000 ≧1 NEB4 100% ≧32000   25% 32 ≧1000 MluI-HF performed best in NEB2 and NEB4, inwhich the FI was ≧32000; MluI-WT performed best in NEB3, in which the FIwas 2000. The overall improvement factor is ≧32000/2000 = ≧16.

Example 29 Engineering of HF BanI 1. Expression of BanI

BanI was expressed in E. coli transformed with pUC19-BanIR andpACYC184-BanIM, each contains BanI endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of BanI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 8, 9, 11, 12, 14, 15, 16, 19, 22,23, 27, 28, 29, 30, 31, 32, 33, 36, 37, 40, 41, 42, 43, 47, 50, 52, 53,54, 55, 56, 58, 61, 64, 66, 67, 69, 70, 71, 75, 76, 81, 82, 84, 85, 86,87, 89, 90, 92, 93, 94, 96, 97, 100, 103, 105, 106, 107, 109, 110, 111,112, 114, 115, 117, 121, 122, 123, 124, 126, 130, 131, 133, 135, 136,138, 139, 140, 141, 143, 145, 146, 148, 150, 151, 152, 154, 156, 157,160, 161, 169, 171, 174, 175, 176, 178, 179, 182, 183, 185, 187, 188,191, 192, 193, 194, 195, 197, 198, 201, 202, 203, 208, 209, 211, 212,213, 215, 217, 218, 220, 221, 224, 225, 226, 229, 232, 233, 234, 236,237, 238, 240, 242, 243, 244, 245, 246, 248, 249, 251, 252, 253, 254,255, 256, 257, 259, 260, 262, 266, 267, 268, 269, 270, 271, 275, 277,279, 281, 282, 283, 284, 285, 287, 288, 289, 291, 292, 294, 296, 298,301, 302, 303, 304, 305, 312, 313, 315, 316, 318, 319, 320, 321, 324,325, 328, 329, 330, 331, 333, 337, 338, 339, 340, 342, 346; Tyr waschanged to Phe at the positions of 104, 125, 127, 156, 159, 204, 239,297, 306, and 336.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2683.

3. Selection of BanI-HF

Selection of BanI-HF was achieved using comparison of activity in waterand NEB4 versus glycerol and NEB ExoI buffer using lambda DNA assubstrate. Mutants with as much or more activity than WT in NEB4 wereselected if they also showed a change in star activity when tested underglycerol conditions. Another indicator used in selecting these mutantswas the fact that removing star activity creates a slow site in cognatecleavage. Numerous mutants were found to have changes in star activityand the resulting slow site: N016A, S33A, P36A, H76A, P87A, N89A, R90A,T138A, K141A, K143A, Q221A, Q224A, N253A, Q292A, R296A, T152I, G326A,and T324A. BanI(Q292A) is designated as BanI-HF.

4. Purification of BanI-HF

Two liters of cell ER2683(pUC19-BanI(P154A), pACYC184-BanIM)) were grownin LB with 100 μg/ml Amp and 33 μg/ml Cam at 37° C. overnight. The cellswere harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mMNaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by is thesame buffer by syringe injection. The column was then loaded on thesystem by the following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mMNaCl, 100 ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient andfollowed by a 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The elutedfractions were then tested for activity. The fractions with highestactivity were further concentrated by Vivaspin® 15R (Vivascience, nowSartorius Vivascience GmbH, Goettingen, Germany). The concentratedBanI-HF was then added an equal volume of glycerol and stored at −20° C.

5. Comparison of BanI-HF and BanI-WT

The FIs of BanI-HF and WT BanI have been determined separately on lambdaDNA in four NEB buffers with diluent A. The result is listed in Table 30(below).

TABLE 30 Comparison of BanI-HF and BanI-WT BanI-HF BanI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1   50% ≧1000   25%  4 ≧250NEB2 12.5% ≧250   25%  4 ≧63 NEB3  0.4% ≧8  6.3%  2 ≧4 NEB4  100% ≧2000 100% 16 ≧125 BanI-HF performed best in NEB4, in which the FI was ≧2000;WT BanI also performed best in NEB4, but the FI was only 16. The overallimprovement factor is ≧2000/16 = ≧125.

Example 30 Engineering of HF KasI 1. Expression of KasI

KasI was expressed in E. coli transformed with placZZ-KasIR andpACY-SfoIM, each contains KasI endonuclease and methylase gene. Thecells were grown at 30° C. overnight in LB with Amp and Cam.

2. Mutagenesis of KasI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 8, 9, 11, 13, 14, 17, 18, 21, 24,26, 28, 29, 31, 32, 33, 34, 36, 37, 39, 42, 43, 44, 47, 48, 51, 52, 54,55, 56, 58, 60, 62, 63, 64, 65, 66, 69, 70, 73, 76, 77, 78, 79, 83, 85,86, 88, 89, 90, 91, 92, 93, 94, 98, 100, 101, 102, 103, 104, 108, 110,111, 114, 115, 116, 117, 118, 119, 122, 123, 124, 125, 126, 128, 129,134, 137, 138, 139, 140, 142, 143, 144, 145, 146, 149, 150, 152, 153,154, 156, 158, 161, 162, 163, 164, 165, 167, 168, 173, 177, 178, 180,181, 182, 184, 185, 188, 189, 190, 191, 192, 195, 197, 198, 200, 202,203, 204, 210, 211, 212, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 225, 226, 228, 229, 231, 234, 237, 238, 241, 243, 244, 245, 246,248, 251, 253, 255, 257, 258, 259, 260, 261, 263, 264, 265, 266, 269,270, 271, 274, 275, 276, 277, and 278; Tyr was changed to Phe at thepositions of 19, 41, 74, 80, 95, 207, and 256.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2683.

3. Selection of KasI-HF

Selection of KasI-HF was achieved using comparison of activity in NEB3and NEB4 using pBR322 DNA as substrate. Mutants with more activity thanWT in NEB4 were selected as increased activity in NEB4 is an indicatorof improved fidelity. The following mutants were found to have moreactivity in NEB4: K024A, P214A, E146A, N251A and Y095F. KasI(N251A) isdesignated as KasI-HF.

4. Purification of KasI-HF

Two liters of cell ER2683(pLacZZ-KasI(M251A), pACYC-SfoIM)) were grownin LB with 100 μg/ml Amp and 33 μg/ml Cam at 30° C. overnight. The cellswere harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mMNaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated KasI-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of KasI-HF and KasI-WT

The FIs of KasI-HF and KasI-WT have been determined separately on pBR322DNA in four NEB buffers with diluent B. The result is listed in Table 31(below).

TABLE 31 Comparison of KasI-HF and KasI-WT KasI-HF KasI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1   50% ≧8000 100% 1 ≧8000 NEB2 100% ≧16000 100% 8 ≧2000 NEB3 12.5% ≧2000 100% 8 ≧250 NEB4  100% ≧16000100% 4 ≧4000 KasI-HF performed best in NEB2 and NEB4, in which the FI is≧16000; KasI-WT performed same in all buffers, in which the best FI is8. The overall improvement factor is 16000/8 = ≧2000.

Example 31 Engineering of HF NruI 1. Expression of NruI

NruI was expressed in E. coli transformed with pUC19-NruIR andpACYC-Sbo13IM, each contains NruI endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of NruI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 8, 10, 12, 13, 15, 16, 19, 20, 21, 22,23, 25, 26, 30, 34, 36, 38, 39, 44, 45, 46, 47, 49, 50, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 68, 70, 71, 72, 73, 75, 77, 79, 80, 82,83, 84, 85, 87, 89, 90, 91, 92, 93, 95, 96, 97, 99, 101, 103, 104, 106,107, 112, 113, 114, 115, 117, 118, 119, 124, 125, 127, 132, 134, 137,138, 139, 141, 146, 147, 148, 149, 152, 154, 155, 157, 158, 159, 162,163, 165, 166, 168, 169, 170, 171, 174, 175, 177, 178, 180, 182, 184,186, 188, 189, 190, 191, 193, 196, 197, 200, 201, 202, 204, 205, 206,207, 208, 209, 211, and 213; Tyr was changed to Phe at the positions of11, 31, 52, 69, 98, 64, and 187.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2683.

3. Selection of NruI-HF

Selection of NruI-HF was achieved using comparison of activity in NEB3and NEB4 using dam− lambda DNA as substrate. Mutants with more activitythan WT in NEB4 were selected as increased activity in NEB4 is anindicator of improved fidelity. The following mutants were found to haveis more activity in NEB4: G075A, Q099A, G155A, and P022A/R90A. P154ANruI(P022A/R90A) is designated as NruI-HF.

4. Purification of NruI-HF

Two liters of cell ER2683(pUC19-NruI(P022AR90A), pACYC184-Sbo13IM) weregrown in LB with 100 μg/ml Amp and 33 μg/ml Cam at 37° C. overnight. Thecells were harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50mM NaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated NruI-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of NruI-HF and NruI-WT

The FIs of NruI-HF and NruI-WT have been determined separately on dam−lambda DNA in four NEB buffers with diluent A. The result is listed inTable 32 (below).

TABLE 32 Comparison of NruI-HF and NruI-WT NruI-HF NruI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1  0.4% ≧64 12.5%  64 ≧1 NEB2 6.3% ≧1000   50% 250 ≧4 NEB3  6.3% ≧1000  100% 500 ≧2 NEB4  100% ≧1600012.5%  32 ≧32 NruI-HF performed best in NEB4, in which the FI was≧16000; NruI-WT performed best in NEB3, in which the FI was 500. Theoverall improvement factor is ≧16000/500 = ≧32.

Example 32 Engineering of High Fidelity NspI 1. Expression of NspI

NspI was expressed in E. coli transformed with pUC19-NspIR andpACYC-FatIM, each contains NspI endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of NspI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 9, 10, 12, 13, 14, 16, 17, 18, 19, 20,21, 23, 26, 29, 30, 31, 32, 34, 36, 37, 39, 40, 41, 42, 44, 45, 46, 47,50, 51, 52, 53, 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 70, 71, 72,73, 74, 77, 78, 80, 81, 82, 83, 85, 86, 87, 89, 90, 91, 93, 94, 96, 97,99, 100, 102, 104, 107, 108, 111, 114, 116, 117, 120, 121, 122, 123,124, 125, 126, 127, 128, 132, 133, 134, 136, 138, 139, 141, 143, 144,145, 146, 147, 149, 150, 152, 153, 154, 155, 157, 158, 159, 161, 164,165, 166, 167, 168, 169, 170, 171, 172, 175, 176, 177, 178, 180, 181,184, 185, 186, 187, 188, 189, 191, 193, 195, 199, 200, 201, 202, 203,205, 206, 208, 209, 210, 211, 212, 213, 215, 216, 217, 220, 222, 225,227, 230, 231, 234, 235, 236, and 238; Tyr was changed to Phe at thepositions of 48, 75, 113, 115, 198, and 224.

The mutagenesis methods were inverse PCR with paired primers is followedby DpnI digestion. The treated product was then transformed into E. colistrain ER2566.

3. Selection of NspI-HF

Selection of NspI-HF was achieved using comparison of activity in NEB3and NEB4 using pBR322 DNA as substrate. Mutants with more activity thanWT in NEB4 were selected as increased activity in NEB4 is an indicatorof improved fidelity. The following mutants were found to have moreactivity in NEB4: S097A and E125A. NspI(S097A) is designated as NspI-HF.

4. Purification of NspI-HF

Two liters of cell ER2566(pUC19-NspI(S097A), pACYC-FatIM)) were grown inLB with 100 μg/ml Amp and 33 μg/ml Cam at 37° C. overnight. The cellswere harvested and sonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mMNaCl. After a centrifugation at 15,000 rpm for 30 minutes, thesupernatant was loaded on the 5 ml HiTrap™ Heparin HP column (GEHealthcare, now Pfizer, Inc., Piscataway, N.J.) pre-balanced by the samebuffer by syringe injection. The column was then loaded on the system bythe following procedure: 48 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100ml 10 mM Tris-HCl, pH 7.5, 50 mM-1M NaCl linear gradient and followed bya 10 ml 10 mM Tris-HCl, pH 7.5, 1M NaCl step. The eluted fractions werethen tested for activity. The fractions with highest activity werefurther concentrated by Vivaspin® 15R (Vivascience, now SartoriusVivascience GmbH, Goettingen, Germany). The concentrated NspI-HF wasthen added an equal volume of glycerol and stored at −20° C.

5. Comparison of NspI-HF and NspI-WT

The FIs of NspI-HF and NspI-WT have been determined separately on pUC19DNA in four NEB buffers with diluent A with BSA. The comparison is shownin FIG. 16, and the result is listed in Table 33 (below).

TABLE 33 Comparison of NspI-HF and NspI-WT NspI-HF NspI-WT ImprovementBuffer Activity FI Activity FI Factor NEB1  100% ≧4000 100% 250 ≧16 NEB2 100% ≧500 100% 16 ≧32 NEB3 12.5% ≧250  25% 120 ≧50 NEB4  100%  500  50%32 ≧16 NspI-HF performed best in NEB1 and NEB4, in which the best FI was≧4000; WT NspI performed best in NEB1 and NEB2, in which the best FI was250. The overall improvement factor is ≧4000/250 = ≧16.

Example 33 Engineering of HF BsrFI 1. Expression of BsrFI

BsrFI was expressed in E. coli transformed with pBAD-BsrFIR andpSYX33-HpaIIM, each contains BsrFI endonuclease and methylase gene. Thecells were grown at 37° C. overnight in LB with Amp and Kan witharabinose induction.

2. Mutagenesis of BsrFI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 8, 9, 12, 13, 15, 16, 17, 18, 19,20, 21, 23, 25, 26, 28, 32, 35, 36, 37, 39, 40, 41, 42, 44, 45, 46, 48,49, 51, 52, 56, 59, 61, 62, 64, 65, 66, 68, 72, 73, 74, 75, 76, 77, 80,86, 87, 89, 91, 93, 94, 95, 97, 98, 103, 105, 106, 108, 109, 111, 113,114, 117, 118, 119, 120, 121, 122, 123, 126, 128, 129, 130, 133, 134,135, 136, 137, 139, 142, 143, 144, 145, 146, 151, 152, 153, 154, 157,158, 159, 161, 162, 163, 165, 166, 168, 169, 170, 171, 173, 174, 177,180, 181, 183, 184, 185, 187, 189, 190, 194, 196, 198, 199, 200, 202,203, 204, 205, 206, 208, 211, 212, 213, 214, 217, 218, 222, 224, 226,229, 230, 231, 233, 235, 238, 240, 241, 242, 243, 245, 246, 248, 249,250, 253, 254, 257, 258, 259, 262, 264, 265, 266, 267, 268, 269, 272,273, 276, 278, 279, 281, 282, 284, and 285; Tyr is changed to Phe at thepositions of 14, 34, 53, 90, 96, 99, 125, 160, 227, 236, 237.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER2566.

3. Selection of BsrFI-HF

Selection of BsrFI-HF was achieved using comparison of activity in NEB3and NEB4 using pBR322 DNA as substrate. Mutants with more activity thanWT in NEB4 were selected as increased activity in NEB4 is an indicatorof improved fidelity. The following mutants were found to have moreactivity in NEB4: K021A/I031R and T120A. BsrFI(K021A/I031R) isdesignated as BsrFI-HF.

4. Purification of BsrFI-HF

Two liters of cell ER2566(pBAD-BsrFI(K021A/I031R), pSYX33-HpaIIM) weregrown in LB with 100 μg/ml Amp and 33 μg/ml Kan at 37° C. overnight with0.2% arabinose induction after 8 hours. The cells were harvested andsonicated in 20 ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl. After acentrifugation at 15,000 rpm for 30 minutes, the supernatant was loadedon the 5 ml HiTrap™ Heparin HP column (GE Healthcare, now Pfizer, Inc.,Piscataway, N.J.) pre-balanced by the same buffer by syringe injection.The column was then loaded on the system by the following procedure: 48ml 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100 ml 10 mM Tris-HCl, pH 7.5, 50mM-1M NaCl linear gradient and followed by a 10 ml 10 mM Tris-HCl, pH7.5, 1M NaCl step. The eluted fractions were then tested for activity.The fractions with highest activity were further concentrated byVivaspin® 15R (Vivascience, now Sartorius Vivascience GmbH, Goettingen,Germany). The concentrated BsrFI-HF was then added an equal volume ofglycerol and stored at −20° C.

5. Comparison of BsrFI-HF and WT BsrFI

The FIs of BsrFI-HF and BsrFI-WT have been determined separately onpBR322 DNA in four NEB buffers with diluent A. The comparison is shownin FIG. 17, and the result is listed in Table 35 (below).

TABLE 35 Comparison of BsrFI-HF and BsrFI-WT BsrFI-HF BsrFI-WTImprovement Buffer Activity FI Activity FI Factor NEB1  100% ≧500   25%16 ≧32 NEB2 12.5% ≧64  100%  4 ≧500 NEB3 NC NC  3.1%  8 ≧−8 NEB4  100%≧500   50% 16 ≧32 BsrFI-HF performed best in NEB1 and NEB4, in which theFI was ≧500; BsrFI-WT performed best in NEB2, in which the FI was 4. Theoverall improvement factor is ≧500/4 = ≧120.

Example 34 Engineering of HF BspEI 1. Expression of BspEI (SEQ ID No.34)

BspEI was expressed in E. coli transformed with pLazz1-BspEIR andpACYC184-BspEIM, each contains BspEI endonuclease and methylase gene.The cells were grown at 37° C. overnight in LB with Amp and Cam.

2. Mutagenesis of BspEI-HF

All residues Cys, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser, Thrwere changed to Ala at positions 7, 8, 10, 11, 12, 13, 14, 17, 19, 20,21, 22, 23, 27, 30, 31, 33, 34, 35, 36, 37, 39, 42, 43, 44, 45, 46, 48,49, 51, 52, 53, 54, 55, 56, 58, 59, 60, 62, 63, 64, 66, 67, 68, 71, 72,73, 74, 75, 78, 79, 81, 82, 84, 85, 88, 89, 91, 92, 93, 94, 95, 96, 98,101, 102, 103, 106, 107, 108, 110, 111, 113, 114, 115, 117, 121, 122,124, 126, 127, 128, 129, 132, 133, 135, 136, 137, 138, 140, 141, 148,149, 151, 153, 155, 156, 157, 160, 162, 164, 166, 167, 168, 169, 172,174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 189, 192, 193,195, 196, 197, 198, 199, 200, 201, 203, 204, 208, 209, 212, 213, 214,216, 217, 218, 219, 221, 222, 228, 229, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 242, 244, 245, 246, 250, 251, 253, 254, 255, 256,258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 275, 276,277, 281, 282, 283, 285, 286, 288, 289, 293, 294.

The mutagenesis methods were inverse PCR with paired primers followed byDpnI digestion. The treated product was then transformed into E. colistrain ER3081.

3. Selection of BspEI-HF

Selection of BspEI-HF was achieved using comparison of activity in NEB3and NEB4 using unmethylated lambda (λ⁻) DNA as substrate. WT BspEI hasmore activity in NEB3, the one with more activity in NEB4 were selected.6 mutants are found to have more activity in NEB4: K7A, T10A, N11A,N14A, Q232A and T199A. T199A has much higher activity than WT in NEB4.BspEI(T199A) is designated as BspEI-HF.

Example 35 Engineering of High Fidelity BamHI (Additional Mutants)

BamHI (SEQ ID No. 35) recognizes and digests at G/GATCC as described inExample 1 of International Publication No. WO 2009/009797. A mutantBamHI(E163A/E167T) was selected as the high fidelity version of theBamHI.

A complete coverage of mutation was done on BamHI. Aside from theresidues reported in the previous patents and applications, the rest ofthe residues were also mutated to Ala at position of 3, 7, 8, 15, 16,21, 22, 23, 24, 27, 29, 31, 33, 34, 35, 37, 38, 39, 45, 47, 48, 49, 53,54, 55, 56, 57, 58, 59, 60, 63, 64, 67, 68, 73, 74, 79, 80, 82, 83, 85,90, 91, 92, 93, 95, 99, 100, 102, 105, 108, 109, 110, 112, 115, 116,117, 124, 125, 127, 128, 129, 130, 131, 134, 136, 138, 140, 141, 142,143, 144, 145, 147, 148, 151, 152, 156, 158, 159, 162, 164, 166, 168,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 185, 187, 188, 189,190, 191, 192, 194, 197, 198, 203, 206, 210 and 212.

Among these mutants, P92A, P144A, G197A and M198A have higher fidelitythan the wild type BamHI. P92A can be an alternative high fidelityBamHI.

What is claimed is: 1-74. (canceled)
 75. A composition comprising avariant MluI restriction endonuclease having reduced star activity,wherein the variant MluI restriction endonuclease comprises an aminoacid sequence that differs from the amino acid sequence of the parentMluI restriction endonuclease by two amino acid substitutions atpositions corresponding to positions 112 and 132 of SEQ ID NO:28.
 76. Acomposition according to claim 75, wherein the amino acid substitutionsare E112A and R132A.
 77. A DNA encoding the variant of claim 75.